Chimeric antigen receptors targeting epidermal growth factor receptor variant iii

ABSTRACT

The invention provides Chimeric Antigen Receptors (CARs) that specifically bind to EGFRvIII (Epidermal Growth Factor Receptor Variant III). The invention further relates to engineered immune cells comprising such CARs, CAR-encoding nucleic acids, and methods of making thereof, engineered immune cells, and nucleic acids. The invention further relates to therapeutic methods for using these CARs and engineered immune cells for the treatment of EGFRvIII-mediated pathologies, including cancers such as glioblastoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/402,760 filed Jan. 10, 2017, which claims the benefit of U.S.Provisional Application No. 62/431,758 filed Dec. 8, 2016, and U.S.Provisional Application No. 62/281,533 filed Jan. 21, 2016, all of whichare hereby incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled“ALGN_006_03US_SeqList_ST25.txt” created on Jan. 23, 2019 and having asize of 206 KB. The sequence listing contained in this .txt file is partof the specification and is incorporated herein by reference in itsentirety.

FIELD

The invention relates to chimeric antigen receptors (CAR). CARs are ableto redirect immune cell specificity and reactivity toward a selectedtarget exploiting the ligand-binding domain properties. In particular,the invention relates to CARs that specifically bind to Epidermal GrowthFactor Receptor Variant III (EGFRvIII specific CARs). The inventionfurther relates to polynucleotides encoding EGFRvIII specific CAR andisolated cells expressing EGFRvIII specific CARs at their surface. Theinvention further relates to methods for engineering immune cellsexpressing EGFRvIII specific CARs at their surface. The invention isparticularly useful for the treatment of solid tumors such asglioblastoma multiforme (GBM), non-small cell lung cancer, head and neckcancer, breast cancer, ovarian cancer, and prostate cancer. Theinvention further relates to immune cells comprising the EGFRvIIIspecific CARs (EGFRvIII specific CAR-T cells), compositions comprisingthe EGFRvIII specific CAR-T cells, and methods of using the EGFRvIIIspecific CAR-T cells for treating EGFRvIII-mediated pathologies.

BACKGROUND

EGFR variant III (EGFRvIII), a tumor specific mutant of EGFR, is aproduct of genomic rearrangement which is often associated withwild-type EGFR gene amplification. EGFRvIII is formed by an in-framedeletion of exons 2-7, leading to deletion of 267 amino acids with aglycine substitution at the junction. The truncated receptor loses itsability to bind ligands but acquires constitutive kinase activity.Interestingly, EGFRvIII always co-expresses with full length wild-typeEGFR in the same tumor cells. Moreover, EGFRvIII expressing cellsexhibit increased proliferation, invasion, angiogenesis and resistanceto apoptosis.

EGFRvIII is most often found in glioblastoma multiforme (GBM). It isestimated that 25-35% of GBM carries this truncated receptors. Moreover,its expression often reflects a more aggressive phenotype and poorprognosis. Besides GBM, expression of EGFRvIII has also been reported inother solid tumors such as non-small cell lung cancer, head and neckcancer, breast cancer, ovarian cancer and prostate cancer. In contrast,EGFRvIII is not expressed in healthy tissues. The lack of expression innormal tissues makes EGFRvIII an ideal target for developing tumorspecific targeted therapy.

Adoptive transfer of T cells genetically modified to recognizemalignancy-associated antigens has shown promise as a new approach totreating cancer (see, e.g., Brenner et al., Current Opinion inImmunology, 22(2): 251-257 (2010); Rosenberg et al., Nature ReviewsCancer, 8(4): 299-308 (2008)). T cells can be genetically modified toexpress chimeric antigen receptors (CARs), which are fusion proteinscomprised of an antigen recognition moiety and T cell activation domains(see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724(1993), and Sadelain et al., Curr. Opin. Immunol, 21(2): 215-223(2009)). Accordingly, treatment to a solid tumor such as glioblastomamultiforme using an anti-EGFRvIII antagonist including EGFRvIII specificCARs and EGFRvIII specific CAR-T cells would make a promisingtherapeutic agent.

SUMMARY

Chimeric antigen receptors (CARs) that bind to EGFRvIII are provided. Itis demonstrated that certain EGFRvIII specific CARs are effective whenexpressed in T cells to activate T cells upon contact with EGFRvIII.Advantageously, the EGFRvIII specific CARs provided herein bind humanEGFRvIII. Also advantageously, the EGFRvIII specific CAR-T cellsprovided herein exhibit degranulation activity, increased interferongamma production, and/or cytotoxic activity upon contact withEGFRvIII-expressing cells.

In one aspect, the invention provides an EGFRvIII specific CARcomprising an extracellular ligand-binding domain, a first transmembranedomain, and an intracellular signaling domain, wherein the extracellularligand-binding domain comprises (a) a heavy chain variable (VH) regioncomprising (i) a VH complementarity determining region one (CDR1)comprising the sequence shown in SEQ ID NO: 62, 63, 64, 74, 75, 76, 80,81, 82, 88, 89, 90, 109, 110, 111, 115, 116, 117, 121, 122, 123, 137,138, or 139; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO:70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 112, 113, 118, 119, 124, 125,127, 128, 140, or 141; and iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 73, 79, 85, 114, 120, 126, 129, or 142, and/or (b) a lightchain variable (VL) region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 149, 156, 159, 162, 165, 182, 185, 187, or195; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 152,157, 160, 163, 183, 186, 188, or 196; and (iii) a VL CDR3 comprising thesequence shown in SEQ ID NO: 153, 158, 161, 164, 184, 189, or 197.

In another aspect, the invention provides an EGFRvIII specific CARcomprising an extracellular ligand-binding domain, a first transmembranedomain, and an intracellular signaling domain, wherein the extracellularligand-binding domain comprises a single chain Fv fragment (scFv)comprising a heavy chain variable (VH) region comprising three CDRs fromthe VH region comprising the sequence shown in SEQ ID NO: 5, 9, 11, 13,15, 37, 39, 41, 43, or 48; and/or a light chain variable (VL) regioncomprising three CDRs from the VL region comprising the sequence shownin SEQ ID NO: 6, 10, 12, 14, 16, 38, 40, 42, or 49. In some embodiments,the VH region can comprise the sequence shown in SEQ ID NO: 5, 9, 11,13, 15, 37, 39, 41, 43, or 48, or a variant thereof with one or severalconservative amino acid substitutions in residues that are not within aCDR and/or the VL region can comprise the amino acid sequence shown inSEQ ID NO: 6, 10, 12, 14, 16, 38, 40, 42, or 49, or a variant thereofwith one or several amino acid substitutions in amino acids that are notwithin a CDR. For example, in some embodiments, the VH or VL region ofthe scFv can comprise an amino acid sequence described above or avariant thereof with no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1conservative substitutions in residues that are not within a CDR.

In some embodiments, the invention provides an EGFRvIII specific CARcomprising an extracellular ligand-binding domain, a first transmembranedomain, and an intracellular signaling domain, wherein the extracellularligand-binding domain comprises a single chain Fv fragment (scFv)comprising a heavy chain variable (VH) region comprising the sequenceshown in SEQ ID NO: 11, 15, 30, 37, or 41; and/or a light chain variable(VL) region comprising the sequence shown in SEQ ID NO: 12, 16, 31, 38,or 42. In some embodiments, the VH comprises the sequence shown in SEQID NO: 11 and the VL comprises the sequence shown in SEQ ID NO: 12. Insome embodiments, the VH comprises the sequence shown in SEQ ID NO: 15and the VL comprises the sequence shown in SEQ ID NO: 16. In someembodiments, the VH comprises the sequence shown in SEQ ID NO: 30 andthe VL comprises the sequence shown in SEQ ID NO: 31. In someembodiments, the VH comprises the sequence shown in SEQ ID NO: 37 andthe VL comprises the sequence shown in SEQ ID NO: 38. In someembodiments, the VH comprises the sequence shown in SEQ ID NO: 41 andthe VL comprises the sequence shown in SEQ ID NO: 42.

In some embodiments, the intracellular signaling domain comprises aCD3zeta signaling domain. In some embodiments, the intracellularsignaling domain comprises a 4-1BB signaling domain. In someembodiments, the CAR can further comprise a second intracellularsignaling domain. In some embodiments, the second intracellularsignaling domain can comprise a 4-1BB signaling domain. In someembodiments the first intracellular signaling domain comprises a CD3zetasignaling domain and the second intracellular signaling domain comprisesa 4-1BB signaling domain.

In some embodiments, the CAR can comprise a stalk domain between theextracellular ligand-binding domain and the first transmembrane domain.In some embodiments, the stalk domain can be selected from the groupconsisting of: a human CD8a hinge, a human CD28 hinge, an IgG1 hinge,and an FcγRIIIα hinge.

In some embodiments, the first transmembrane domain can comprise a CD8achain transmembrane domain.

In some embodiments, the CAR can comprise another extracellularligand-binding domain which is not specific for EGFRvIII.

In some embodiments of a CAR, the extracellular ligand-bindingdomain(s), the first transmembrane domain, and intracellular signalingdomain(s) are on a single polypeptide.

In some embodiments, the CAR can comprise a second transmembrane domain,wherein the first transmembrane domain and the extracellularligand-binding domain(s) are on a first polypeptide, and wherein thesecond transmembrane domain and the intracellular signaling domain(s)are on a second polypeptide, wherein the first transmembrane domaincomprises a transmembrane domain from the α chain of the high-affinityIgE receptor (FcεRI) and the second transmembrane domain comprises atransmembrane domain from the γ or β chain of FcεRI. In someembodiments, the CAR can comprise a third polypeptide comprising a thirdtransmembrane domain fused to an intracellular signaling domain from aco-stimulatory molecule, wherein the third transmembrane domaincomprises a transmembrane domain from the γ or β chain of FcεRI.

In another aspect, the invention provides an isolated polynucleotidecomprising a nucleic acid sequence encoding an EGFRvIII specific CAR asdescribed herein.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid sequence encoding an EGFRvIII specific CARantibody as described herein.

In another aspect, the invention provides an engineered immune cellexpressing at its cell surface membrane an EGFRvIII specific CAR asdescribed herein. In some embodiments, the engineered immune cell cancomprise another CAR which is not specific for EGFRvIII.

In some embodiments, the engineered immune cell can comprise apolynucleotide encoding a suicide polypeptide. In some embodiments, thesuicide polypeptide is RQR8. In some embodiments, the polynucleotideencoding the suicide polypeptide is in a different nucleic acid moleculethan the polynucleotide comprising a nucleic acid sequence encoding theEGFRvIII specific CAR. In some embodiments, the polynucleotide encodingthe suicide polypeptide is part of the same nucleic acid molecule as thepolynucleotide comprising a nucleic acid sequence encoding the EGFRvIIIspecific CAR.

In some embodiments, an engineered immune cell containing anEGFRvIII-specific CAR can comprise a suicide polypeptide in a separatepolypeptide chain from the polypeptide chain of the EGFRvIII-specificCAR.

In some embodiments, an EGFRvIII specific CAR as described herein alsocomprises a suicide polypeptide in the same polypeptide chain as theCAR. For example, the suicide polypeptide may be between the scFv andhinge sequence of the CAR. In some embodiments, a suicide polypeptide ina CAR may have the R2 format as provided herein. In some embodiments, asuicide polypeptide comprises an epitope that is recognized byrituximab.

Also provided herein is a polynucleotide encoding an EGFRvIII specificCAR which also encodes a suicide polypeptide in the CAR.

In some embodiments, an engineered immune cell can be derived from aninflammatory T-lymphocyte, a cytotoxic T-lymphocyte, a regulatoryT-lymphocyte, a memory T-lymphocyte, a helper T-lymphocyte, a naturalkiller T-lymphocyte, or a natural killer cell.

In some embodiments, the engineered immune cell can comprise adisruption in one or more endogenous genes, wherein the endogenous geneencodes TCRα, TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidinekinase (dCK), or an immune checkpoint protein such as for exampleprogrammed death-1 (PD-1).

In some embodiments, the immune cell is obtained from a healthy donor.In some embodiments, the immune cell is obtained from a patient.

In another aspect, the invention provides an engineered immune cellexpressing at its cell surface membrane an EGFRvIII specific CAR asdescribed herein for use as a medicament. In some embodiments, themedicament is for use in treatment of an EGFRvIII related cancer (e.g.,any cancer with EGFRvIII expression) selecting from the group consistingof glioblastoma multiform, anaplastic astrocytoma, giant cellglioblastoma, gliosarcoma, anaplastic oligodendroglioma, anaplasticependymoma, anaplastic oligoastrocytoma, choroid plexus carcinoma,anaplastic ganglioglioma, pineoblastoma, pineocytoma, meningioma,medulloepithelioma, ependymoblastoma, medulloblastoma, supraentorialprimitive neuroectodermal tumor, atypical teratoid/rhabdoid tumor, mixedglioma, head and neck cancer, non-small cell lung cancer, breast cancer,ovarian cancer, prostate cancer, medullobastoma, colorectal cancer, analcancer, cervical cancer, renal cancer, skin cancer, pancreatic cancer,liver cancer, bladder cancer, gastric cancer, thyroid cancer,mesothelioma, uterine cancer, lymphoma, and leukemia.

In another aspect, the invention provides a method of engineering animmune cell comprising: providing an immune cell; and expressing at thesurface of the cell at least one EGFRvIII specific CAR as describedherein.

In some embodiments, the method comprises: providing an immune cell;introducing into the cell at least one polynucleotide encoding saidEGFRvIII specific CAR; and expressing said polynucleotide into the cell.

In some embodiments, the method comprises providing an immune cell;introducing into the cell at least one polynucleotide encoding saidEGFRvIII specific CAR; and introducing at least one other CAR which isnot specific for EGFRvIII.

In another aspect, the invention provides a method of treating a subjectsuffering from a condition associated with malignant cells, the methodcomprising: providing an immune cell expressing at the surface anEGFRvIII specific CAR as described herein; and administering said immunecells to said patient.

In another aspect, the invention provides a pharmaceutical compositioncomprising an engineered immune cell as described herein.

In another aspect, the invention provides a method of treating acondition associated with malignant cells expressing EGFRvIII in asubject comprising administering to a subject in need thereof aneffective amount of a pharmaceutical composition of claim comprising anengineered immune cell as described herein. In some embodiments, thecondition is a cancer. In some embodiments, the cancer is an EGFRvIIIrelated cancer selecting from the group consisting of glioblastomamultiform, anaplastic astrocytoma, giant cell glioblastoma, gliosarcoma,anaplastic oligodendroglioma, anaplastic ependymoma, anaplasticoligoastrocytoma, choroid plexus carcinoma, anaplastic ganglioglioma,pineoblastoma, pineocytoma, meningioma, medulloepithelioma,ependymoblastoma, medulloblastoma, supraentorial primitiveneuroectodermal tumor, atypical teratoid/rhabdoid tumor, mixed glioma,head and neck cancer, non-small cell lung cancer, breast cancer, ovariancancer, prostate cancer, medullobastoma, colorectal cancer, anal cancer,renal cancer, cervical cancer, liver cancer, pancreatic cancer, gastriccancer, thyroid cancer, mesothelioma, uterine cancer, and bladdercancer.

In another aspect, the invention provides a method of inhibiting tumorgrowth or progression in a subject who has malignant cells expressingEGFRvIII, comprising administering to the subject in need thereof aneffective amount of a pharmaceutical composition comprising anengineered immune cell as described herein.

In another aspect, the invention provides a method of inhibitingmetastasis of malignant cells expressing EGFRvIII in a subject,comprising administering to the subject in need thereof an effectiveamount of a pharmaceutical composition comprising an engineered immunecell as described herein.

In another aspect, the invention provides a method inducing tumorregression in a subject who has malignant cells expressing EGFRvIII,comprising administering to the subject in need thereof an effectiveamount of a pharmaceutical composition comprising an engineered immunecell as described herein.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIG. 1A, FIG. 1B, and FIG. 1C show examples of FACS binding histogramsof three EGFRvIII antibodies: mAb 42G9 (FIG. 1A), 32A10 (FIG. 1B) and32G8 (FIG. 1C), to three F98 cell lines: F98 (EGFR negative),F98-EGFRwt, and F98-EGFRvIII. The X-axis is fluorescence intensity; theY-axis is percentage of maximum/normalized to mode.

FIG. 2A shows a bar graph summarizing EGFRvIII specific CAR expressionfor CARs containing different EGFRvIII specific clones.

FIG. 2B shows a bar graph summarizing the percentage of EGFRvIIIspecific CAR T cells that bound to recombinant EGFR-mFc andEGFRvIII-mFc.

FIG. 2C shows a bar graph summarizing the expression of CARs containing10 different EGFRvIII specific clones in CAR T cells.

FIG. 3 shows a bar graph summarizing degranulation activities ofEGFRvIII specific CAR T cells expressing different EGFRvIII specificclones, alone or upon co-culture with various cell lines: cells that donot express any EGFR protein (NCI-H522 and U87-KO), cells express highlevel of wild-type EGFR (U87-KO-EGFRwt), or cells that express low(NCI-H522-EGFRvIII) and high (U87-KO-EGFRvIII) levels of EGFRvIII.

FIG. 4 shows a bar graph summarizing IFNγ secretion by EGFRvIII specificCAR T cells expressing different EGFRvIII specific clones, alone or uponco-culture with various cell lines: cells that do not express any EGFRprotein (NCI-H522 and U87-KO), cells express high level of wild-typeEGFR (U87-KO-EGFRwt), or cells that express low (NCI-H522-EGFRvIII) andhigh (U87-KO-EGFRvIII) levels of EGFRvIII.

FIG. 5 shows a bar graph comparing the cytotoxicity of EGFRvIII specificCAR T cells expressing different EGFRvIII specific clones towards awild-type EGFR expressing cell line (U87-KO-EGFRwt) vs high(U87-KO-EGFRvIII) and low EGFRvIII (NCI-H522-EGFRvIII) expressing celllines.

FIG. 6 shows a graph summarizing the anti-tumor activities of EGFRvIIIspecific CAR T cells expressing different EGFRvIII specific clonesagainst GBM cells in a subcutaneous GBM xenograft model.

FIG. 7 shows a bar graph summarizing the CAR expression by CAR T cellsexpressing four different EGFRvIII specific clones in the CAR andcarrying the intra-CAR suicide sequence R2 in the CAR on Day 4, Day9/10, and Day 14/15 post-T cell transduction.

FIG. 8 shows a bar graph summarizing the CAR expression by CAR T cellsexpressing four different EGFRvIII specific clones in the CAR andcarrying the intra-CAR suicide sequence R2 on Day 4 and Day 14/15 post-Tcell transduction.

FIG. 9 shows a bar graph summarizing the cytotoxicity of CAR T cellsexpressing four different EGFRvIII specific clones and carrying theintra-CAR suicide sequence R2 against a high level (U87-KO-EGFRvIII) anda low level EGFRvIII (NCI-H522-EGFRvIII) expressing cell line.

DETAILED DESCRIPTION

The invention disclosed herein provides chimeric antigen receptors(CARs) and immune cells comprising CARs (e.g. CAR-T cells) thatspecifically bind to EGFRvIII (e.g., human EGFRvIII). The invention alsoprovides polynucleotides encoding these CARs, compositions comprisingthese CAR-T cells, and methods of making and using these CARs and CAR-Tcells. The invention also provides methods for treating a conditionassociated with EGFRvIII-mediated pathologies in a subject, such ascancer.

General Techniques

The practice of the invention will employ, unless otherwise indicated,conventional techniques of molecular biology (including recombinanttechniques), microbiology, cell biology, biochemistry, immunology,virology, monoclonal antibody generation and engineering, which arewithin the skill of the art. Such techniques are explained fully in theliterature, such as, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

The term “extracellular ligand-binding domain” as used herein refers toan oligo- or polypeptide that is capable of binding a ligand.Preferably, the domain will be capable of interacting with a cellsurface molecule. For example, the extracellular ligand-binding domainmay be chosen to recognize a ligand that acts as a cell surface markeron target cells associated with a particular disease state.

The term “stalk domain” or “hinge domain” are used interchangeablyherein to refer to any oligo- or polypeptide that functions to link thetransmembrane domain to the extracellular ligand-binding domain. Inparticular, stalk domains are used to provide more flexibility andaccessibility for the extracellular ligand-binding domain.

The term “intracellular signaling domain” refers to the portion of aprotein which transduces the effector function signal and directs thecell to perform a specialized function.

A “co-stimulatory molecule” as used herein refers to the cognate bindingpartner on a T cell that specifically binds with a co-stimulatoryligand, thereby mediating a co-stimulatory response by the cell, suchas, but not limited to proliferation. Co-stimulatory molecules include,but are not limited to an MHC class I molecule, BTLA and Toll ligandreceptor. Examples of costimulatory molecules include CD27, CD28, CD8,4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 anda ligand that specifically binds with CD83 and the like.

A “co-stimulatory ligand” refers to a molecule on an antigen presentingcell that specifically binds a cognate co-stimulatory signal molecule ona T cell, thereby providing a signal which, in addition to the primarysignal provided by, for instance, binding of a TCR/CD3 complex with anMHC molecule loaded with peptide, mediates a T cell response, including,but not limited to, proliferation activation, differentiation, cytokineproduction, and the like. A co-stimulatory ligand can include but is notlimited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L,inducible costimulatory igand (ICOS-L), intercellular adhesion molecule(ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin preceptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Tollligand receptor and a ligand that specifically binds with B7-H3. Aco-stimulatory ligand also encompasses, inter alia, an antibody thatspecifically binds with a co-stimulatory molecule present on a T cell,such as but not limited to, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD1,ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT,NKG2C, B7-H3, a ligand that specifically binds with CD83.

An “antibody” is an immunoglobulin molecule capable of specific bindingto a target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. As used herein, the termencompasses not only intact polyclonal or monoclonal antibodies, butalso antigen binding fragments thereof (such as Fab, Fab′, F(ab′)₂, Fv),single chain (scFv) and domain antibodies (including, for example, sharkand camelid antibodies), and fusion proteins comprising an antibody, andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site. An antibody includes an antibodyof any class, such as IgG, IgA, or IgM (or sub-class thereof), and theantibody need not be of any particular class. Depending on the antibodyamino acid sequence of the constant region of its heavy chains,immunoglobulins can be assigned to different classes. There are fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantregions that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “antigen binding fragment” or “antigen binding portion” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., EGFRvIII). Antigen binding functions of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term “antigen binding fragment” of anantibody include Fab; Fab′; F(ab′)₂; an Fd fragment consisting of the VHand CH1 domains; an Fv fragment consisting of the VL and VH domains of asingle arm of an antibody; a single domain antibody (dAb) fragment (Wardet al., Nature 341:544-546, 1989), and an isolated complementaritydetermining region (CDR).

An antibody, an antibody conjugate, or a polypeptide that“preferentially binds” or “specifically binds” (used interchangeablyherein) to a target (e.g., EGFRvIII protein) is a term well understoodin the art, and methods to determine such specific or preferentialbinding are also well known in the art. A molecule is said to exhibit“specific binding” or “preferential binding” if it reacts or associatesmore frequently, more rapidly, with greater duration and/or with greateraffinity with a particular cell or substance than it does withalternative cells or substances. An antibody “specifically binds” or“preferentially binds” to a target if it binds with greater affinity,avidity, more readily, and/or with greater duration than it binds toother substances. For example, an antibody that specifically orpreferentially binds to an EGFRvIII epitope is an antibody that bindsthis epitope with greater affinity, avidity, more readily, and/or withgreater duration than it binds to other EGFRvIII epitopes ornon-EGFRvIII epitopes. It is also understood that by reading thisdefinition, for example, an antibody (or moiety or epitope) thatspecifically or preferentially binds to a first target may or may notspecifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. As known in the art, the variableregions of the heavy and light chain each consist of four frameworkregions (FR) connected by three complementarity determining regions(CDRs) also known as hypervariable regions. The CDRs in each chain areheld together in close proximity by the FRs and, with the CDRs from theother chain, contribute to the formation of the antigen binding site ofantibodies. There are at least two techniques for determining CDRs: (1)an approach based on cross-species sequence variability (i.e., Kabat etal. Sequences of Proteins of Immunological Interest, (5th ed., 1991,National Institutes of Health, Bethesda Md.)); and (2) an approach basedon crystallographic studies of antigen-antibody complexes (Al-Iazikaniet al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR mayrefer to CDRs defined by either approach or by a combination of bothapproaches.

A “CDR” of a variable domain are amino acid residues within the variableregion that are identified in accordance with the definitions of theKabat, Chothia, the accumulation of both Kabat and Chothia, AbM,contact, and/or conformational definitions or any method of CDRdetermination well known in the art. Antibody CDRs may be identified asthe hypervariable regions originally defined by Kabat et al. See, e.g.,Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, NIH, Washington D.C. The positions of theCDRs may also be identified as the structural loop structures originallydescribed by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, 1989. Other approaches to CDR identification include the“AbM definition,” which is a compromise between Kabat and Chothia and isderived using Oxford Molecular's AbM antibody modeling software (nowAccelrys®), or the “contact definition” of CDRs based on observedantigen contacts, set forth in MacCallum et al., J. Mol. Biol.,262:732-745, 1996. In another approach, referred to herein as the“conformational definition” of CDRs, the positions of the CDRs may beidentified as the residues that make enthalpic contributions to antigenbinding. See, e.g., Makabe et al., Journal of Biological Chemistry,283:1156-1166, 2008. Still other CDR boundary definitions may notstrictly follow one of the above approaches, but will nonethelessoverlap with at least a portion of the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. As used herein, a CDR mayrefer to CDRs defined by any approach known in the art, includingcombinations of approaches. The methods used herein may utilize CDRsdefined according to any of these approaches. For any given embodimentcontaining more than one CDR, the CDRs may be defined in accordance withany of Kabat, Chothia, extended, AbM, contact, and/or conformationaldefinitions.

As used herein, “monoclonal antibody” refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical except forpossible naturally-occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256:495, 1975, or may be madeby recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.The monoclonal antibodies may also be isolated from phage librariesgenerated using the techniques described in McCafferty et al., Nature348:552-554, 1990, for example.

As used herein, “humanized” antibody refers to forms of non-human (e.g.murine) antibodies that are chimeric immunoglobulins, immunoglobulinchains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. Preferably, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementarity determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat, or rabbit having the desiredspecificity, affinity, and capacity. In some instances, Fv frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, the humanized antibodymay comprise residues that are found neither in the recipient antibodynor in the imported CDR or framework sequences, but are included tofurther refine and optimize antibody performance. In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region ordomain (Fc), typically that of a human immunoglobulin. Preferred areantibodies having Fc regions modified as described in WO 99/58572. Otherforms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDRL3, CDR H1, CDR H2, or CDR H3) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody.

As used herein, “human antibody” means an antibody having an amino acidsequence corresponding to that of an antibody produced by a human and/orwhich has been made using any of the techniques for making humanantibodies known to those skilled in the art or disclosed herein. Thisdefinition of a human antibody includes antibodies comprising at leastone human heavy chain polypeptide or at least one human light chainpolypeptide. One such example is an antibody comprising murine lightchain and human heavy chain polypeptides. Human antibodies can beproduced using various techniques known in the art. In one embodiment,the human antibody is selected from a phage library, where that phagelibrary expresses human antibodies (Vaughan et al., NatureBiotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci.(USA) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381,1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies canalso be made by immunization of animals into which human immunoglobulinloci have been transgenically introduced in place of the endogenousloci, e.g., mice in which the endogenous immunoglobulin genes have beenpartially or completely inactivated. This approach is described in U.S.Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and5,661,016. Alternatively, the human antibody may be prepared byimmortalizing human B lymphocytes that produce an antibody directedagainst a target antigen (such B lymphocytes may be recovered from anindividual or from single cell cloning of the cDNA, or may have beenimmunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol.,147 (1):86-95, 1991; and U.S. Pat. No. 5,750,373.

The term “chimeric antibody” is intended to refer to antibodies in whichthe variable region sequences are derived from one species and theconstant region sequences are derived from another species, such as anantibody in which the variable region sequences are derived from a mouseantibody and the constant region sequences are derived from a humanantibody.

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to chains of amino acids of anylength. For example, the chain may be relatively short (e.g., 10-100amino acids), or longer. The chain may be linear or branched, it maycomprise modified amino acids, and/or may be interrupted by non-aminoacids. The terms also encompass an amino acid chain that has beenmodified naturally or by intervention; for example, disulfide bondformation, glycosylation, lipidation, acetylation, phosphorylation, orany other manipulation or modification, such as conjugation with alabeling component. Also included within the definition are, forexample, polypeptides containing one or more analogs of an amino acid(including, for example, unnatural amino acids, etc.), as well as othermodifications known in the art. It is understood that the polypeptidescan occur as single chains or associated chains.

A “monovalent antibody” comprises one antigen binding site per molecule(e.g., IgG or Fab). In some instances, a monovalent antibody can havemore than one antigen binding sites, but the binding sites are fromdifferent antigens.

A “bivalent antibody” comprises two antigen binding sites per molecule(e.g., IgG). In some instances, the two binding sites have the sameantigen specificities. However, bivalent antibodies may be bispecific.

A “bispecific” or “dual-specific” is a hybrid antibody having twodifferent antigen binding sites. The two antigen binding sites of abispecific antibody bind to two different epitopes, which may reside onthe same or different protein targets.

A “bifunctional” is an antibody having identical antigen binding sites(i.e., identical amino acid sequences) in the two arms but each bindingsite can recognize two different antigens.

Antibodies of the invention can be produced using techniques well knownin the art, e.g., recombinant technologies, phage display technologies,synthetic technologies or combinations of such technologies or othertechnologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45:1628-50, 1999 and Fellouse, F. A., et al, J. Mol.Biol., 373(4):924-40, 2007).

As known in the art, “polynucleotide,” or “nucleic acid,” as usedinterchangeably herein, refer to chains of nucleotides of any length,and include DNA and RNA. The nucleotides can be deoxyribonucleotides,ribonucleotides, modified nucleotides or bases, and/or their analogs, orany substrate that can be incorporated into a chain by DNA or RNApolymerase. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thechain. The sequence of nucleotides may be interrupted by non-nucleotidecomponents. A polynucleotide may be further modified afterpolymerization, such as by conjugation with a labeling component. Othertypes of modifications include, for example, “caps”, substitution of oneor more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,carbamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid supports. The 5′ and 3′ terminal OH can be phosphorylated orsubstituted with amines or organic capping group moieties of from 1 to20 carbon atoms. Other hydroxyls may also be derivatized to standardprotecting groups. Polynucleotides can also contain analogous forms ofribose or deoxyribose sugars that are generally known in the art,including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or2′-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. One or more phosphodiesterlinkages may be replaced by alternative linking groups. Thesealternative linking groups include, but are not limited to, embodimentswherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”),(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂ (“formacetal”), in whicheach R or R′ is independently H or substituted or unsubstituted alkyl(1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl,cycloalkyl, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. The preceding description applies toall polynucleotides referred to herein, including RNA and DNA.

As known in the art a “constant region” of an antibody refers to theconstant region of the antibody light chain or the constant region ofthe antibody heavy chain, either alone or in combination.

As used herein, “substantially pure” refers to material which is atleast 50% pure (i.e., free from contaminants), more preferably, at least90% pure, more preferably, at least 95% pure, yet more preferably, atleast 98% pure, and most preferably, at least 99% pure.

A “host cell” includes an individual cell or cell culture that can be orhas been a recipient for vector(s) for incorporation of polynucleotideinserts. Host cells include progeny of a single host cell, and theprogeny may not necessarily be completely identical (in morphology or ingenomic DNA complement) to the original parent cell due to natural,accidental, or deliberate mutation. A host cell includes cellstransfected in vivo with a polynucleotide(s) of this invention.

As used herein, “immune cell” refers to a cell of hematopoietic originfunctionally involved in the initiation and/or execution of innateand/or adaptative immune response.

As known in the art, the term “Fc region” is used to define a C-terminalregion of an immunoglobulin heavy chain. The “Fc region” may be a nativesequence Fc region or a variant Fc region. Although the boundaries ofthe Fc region of an immunoglobulin heavy chain might vary, the human IgGheavy chain Fc region is usually defined to stretch from an amino acidresidue at position Cys226, or from Pro230, to the carboxyl-terminusthereof. The numbering of the residues in the Fc region is that of theEU index as in Kabat. Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991. The Fc region of animmunoglobulin generally comprises two constant regions, CH2 and CH3.

As used in the art, “Fc receptor” and “FcR” describe a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet,Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods,4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995.“FcR” also includes the neonatal receptor, FcRn, which is responsiblefor the transfer of maternal IgGs to the fetus (Guyer et al., J.Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

The term “compete”, as used herein with regard to an antibody, meansthat a first antibody, or an antigen binding fragment (or portion)thereof, binds to an epitope in a manner sufficiently similar to thebinding of a second antibody, or an antigen binding portion thereof,such that the result of binding of the first antibody with its cognateepitope is detectably decreased in the presence of the second antibodycompared to the binding of the first antibody in the absence of thesecond antibody. The alternative, where the binding of the secondantibody to its epitope is also detectably decreased in the presence ofthe first antibody, can, but need not be the case. That is, a firstantibody can inhibit the binding of a second antibody to its epitopewithout that second antibody inhibiting the binding of the firstantibody to its respective epitope. However, where each antibodydetectably inhibits the binding of the other antibody with its cognateepitope or ligand, whether to the same, greater, or lesser extent, theantibodies are said to “cross-compete” with each other for binding oftheir respective epitope(s). Both competing and cross-competingantibodies are encompassed by the invention. Regardless of the mechanismby which such competition or cross-competition occurs (e.g., sterichindrance, conformational change, or binding to a common epitope, orportion thereof), the skilled artisan would appreciate, based upon theteachings provided herein, that such competing and/or cross-competingantibodies are encompassed and can be useful for the methods disclosedherein.

As used herein “autologous” means that cells, a cell line, or populationof cells used for treating patients are originating from said patient orfrom a Human Leucocyte Antigen (HLA) compatible donor.

As used herein “allogeneic” means that cells or population of cells usedfor treating patients are not originating from said patient but from adonor.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing the proliferation of (or destroying) neoplasticor cancerous cells, inhibiting metastasis of neoplastic cells, shrinkingor decreasing the size of EGFRvIII expressing tumor, remission of anEGFRvIII associated disease (e.g., cancer), decreasing symptomsresulting from an EGFRvIII associated disease (e.g., cancer), increasingthe quality of life of those suffering from an EGFRvIII associateddisease (e.g., cancer), decreasing the dose of other medicationsrequired to treat an EGFRvIII associated disease (e.g., cancer),delaying the progression of an EGFRvIII associated disease (e.g.,cancer), curing an EGFRvIII associated disease (e.g., cancer), and/orprolong survival of patients having an EGFRvIII associated disease(e.g., cancer).

“Ameliorating” means a lessening or improvement of one or more symptomsas compared to not administering an EGFRvIII specific CAR.“Ameliorating” also includes shortening or reduction in duration of asymptom.

As used herein, an “effective dosage” or “effective amount” of drug,compound, or pharmaceutical composition is an amount sufficient toeffect any one or more beneficial or desired results. For prophylacticuse, beneficial or desired results include eliminating or reducing therisk, lessening the severity, or delaying the outset of the disease,including biochemical, histological and/or behavioral symptoms of thedisease, its complications and intermediate pathological phenotypespresenting during development of the disease. For therapeutic use,beneficial or desired results include clinical results such as reducingincidence or amelioration of one or more symptoms of various EGFRvIIIassociated diseases or conditions (such as for example glioblastomamultiform), decreasing the dose of other medications required to treatthe disease, enhancing the effect of another medication, and/or delayingthe progression of the EGFRvIII associated disease of patients. Aneffective dosage can be administered in one or more administrations. Forpurposes of this invention, an effective dosage of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective dosage of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective dosage” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

An “individual” or a “subject” is a mammal, more preferably, a human.Mammals also include, but are not limited to primates, horses, dogs,cats, mice and rats.

As used herein, “vector” means a construct, which is capable ofdelivering, and, preferably, expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells.

As used herein, “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid. An expressioncontrol sequence can be a promoter, such as a constitutive or aninducible promoter, or an enhancer. The expression control sequence isoperably linked to the nucleic acid sequence to be transcribed.

As used herein, “pharmaceutically acceptable carrier” or “pharmaceuticalacceptable excipient” includes any material which, when combined with anactive ingredient, allows the ingredient to retain biological activityand is non-reactive with the subject's immune system. Examples include,but are not limited to, any of the standard pharmaceutical carriers suchas a phosphate buffered saline solution, water, emulsions such asoil/water emulsion, and various types of wetting agents. Preferreddiluents for aerosol or parenteral administration are phosphate bufferedsaline (PBS) or normal (0.9%) saline. Compositions comprising suchcarriers are formulated by well known conventional methods (see, forexample, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro,ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Scienceand Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

The term “k_(on)” or “k_(a)”, as used herein, refers to the rateconstant for association of an antibody to an antigen. Specifically, therate constants (k_(on)/k_(a) and k_(off)/k_(d)) and equilibriumdissociation constants may be measured using, for example, full-lengthantibodies and/or Fab antibody fragments and corresponding antigen.

The term “k_(on)” or “k_(d)”, as used herein, refers to the rateconstant for dissociation of an antibody from the antibody/antigencomplex.

The term “K_(D)”, as used herein, refers to the equilibrium dissociationconstant of an antibody-antigen interaction.

Determinations of the association and dissociation rate constants,k_(on) and k_(off) respectively, may be made using a surface plasmonresonance-based biosensor to characterize an analyte/ligand interactionunder conditions where the analyte is monovalent with respect to bindinga ligand that is immobilized at low capacity onto a sensor surface via acapture reagent. The analysis is performed using a kinetic titrationmethodology as described in Karlsson et al., Anal. Biochem 349, 136-147,2006. The sensor chip, capturing reagent, and assay buffer employed fora given assay are chosen to give stable capture of ligand onto thesensor surface, minimize non-specific binding of the analyte to thesurfaces, and yield analyte-binding responses that are appropriate forkinetic analysis, per the recommendations in Myszka, J. Mol. Recognit12, 279-284, 1999. The analyte-binding responses per analyte/ligandinteraction are double referenced and fit to a 1:1 Langmuir “masstransport limited model” with k_(a), k_(d) and R_(max) as globalparameters as described in Myszka & Morton et al., Biophys. Chem 64,127-137 (1997). The equilibrium dissociation constant, K_(D), is deducedfrom the ratio of the kinetic rate constants, K_(D)=k_(off)/k_(on). Suchdeterminations preferably take place at 25° C. or 37° C.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.Generally speaking, the term “about” refers to the indicated value ofthe variable and to all values of the variable that are within theexperimental error of the indicated value (e.g. within the 95%confidence interval for the mean) or within 10 percent of the indicatedvalue, whichever is greater. Where the term “about” is used within thecontext of a time period (years, months, weeks, days etc.), the term“about” means that period of time plus or minus one amount of the nextsubordinate time period (e.g. about 1 year means 11-13 months; about 6months means 6 months plus or minus 1 week; about 1 week means 6-8 days;etc.), or within 10 percent of the indicated value, whichever isgreater.

It is understood that wherever embodiments are described herein with thelanguage “comprising,” otherwise analogous embodiments described interms of “consisting of” and/or “consisting essentially of” are alsoprovided.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Theinvention also envisages the explicit exclusion of one or more of any ofthe group members in the claimed invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent specification, including definitions, will control. Throughoutthis specification and claims, the word “comprise,” or variations suchas “comprises” or “comprising” will be understood to imply the inclusionof a stated integer or group of integers but not the exclusion of anyother integer or group of integers. Unless otherwise required bycontext, singular terms shall include pluralities and plural terms shallinclude the singular.

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the invention. The materials,methods, and examples are illustrative only and not intended to belimiting.

EGFRvIII Specific CARs and Methods of Making Thereof

The invention provides CARs that bind to EGFRvIII (e.g., human EGFRvIII(e.g., SEQ ID NO: 201, accession number: P00533 Feature IdentifierVAR_066493, or GenBank Accession No. AJN69267)). EGFRvIII specific CARsprovided herein include single chain CARS and multichain CARs. The CARshave the ability to redirect T cell specificity and reactivity towardEGFRvIII in a non-MHC-restricted manner, exploiting the antigen-bindingproperties of monoclonal antibodies. The non-MHC-restricted antigenrecognition gives T cells expressing CARs the ability to recognize anantigen independent of antigen processing, thus bypassing a majormechanism of tumor escape.

In some embodiments, CARs provided herein comprise an extracellularligand-binding domain (e.g., a single chain variable fragment (scFv)), atransmembrane domain, and an intracellular signaling domain. In someembodiments, the extracellular ligand-binding domain, transmembranedomain, and intracellular signaling domain are in one polypeptide, i.e.,in a single chain. Multichain CARs and polypeptides are also providedherein. In some embodiments, the multichain CARs comprise: a firstpolypeptide comprising a transmembrane domain and at least oneextracellular ligand-binding domain, and a second polypeptide comprisinga transmembrane domain and at least one intracellular signaling domain,wherein the polypeptides assemble together to form a multichain CAR.

In some embodiments, an EGFRvIII specific multichain CAR is based on thehigh affinity receptor for IgE (FcεRI). The FcεRI expressed on mastcells and basophiles triggers allergic reactions. FcεRI is a tetramericcomplex composed of a single α subunit, a single β subunit, and twodisulfide-linked γ subunits. The α subunit contains the IgE-bindingdomain. The β and γ subunits contain ITAMs that mediate signaltransduction. In some embodiments, the extracellular domain of the FcRαchain is deleted and replaced by an EGFRvIII specific extracellularligand-binding domain. In some embodiments, the multichain EGFRvIIIspecific CAR comprises an scFv that binds specifically to EGFRvIII, theCD8a hinge, and the ITAM of the FcRs chain. In some embodiments, the CARmay or may not comprise the FcRγ chain.

In some embodiments, the extracellular ligand-binding domain comprisesan scFv comprising the light chain variable (VL) region and the heavychain variable (VH) region of a target antigen specific monoclonalantibody joined by a flexible linker. Single chain variable regionfragments are made by linking light and/or heavy chain variable regionsby using a short linking peptide (Bird et al., Science 242:423-426,1988). An example of a linking peptide is the GS linker having the aminoacid sequence (GGGGS)₄ (SEQ ID NO: 202), which bridges approximately 3.5nm between the carboxyl terminus of one variable region and the aminoterminus of the other variable region. Linkers of other sequences havebeen designed and used (Bird et al., 1988, supra). In general, linkerscan be short, flexible polypeptides and preferably comprised of about 20or fewer amino acid residues. Linkers can in turn be modified foradditional functions, such as attachment of drugs or attachment to solidsupports. The single chain variants can be produced either recombinantlyor synthetically. For synthetic production of scFv, an automatedsynthesizer can be used. For recombinant production of scFv, a suitableplasmid containing polynucleotide that encodes the scFv can beintroduced into a suitable host cell, either eukaryotic, such as yeast,plant, insect or mammalian cells, or prokaryotic, such as E. coli.Polynucleotides encoding the scFv of interest can be made by routinemanipulations such as ligation of polynucleotides. The resultant scFvcan be isolated using standard protein purification techniques known inthe art.

In some embodiments, the extracellular ligand-binding domain comprises(a) a VH region comprising (i) a VH complementarity determining regionone (CDR1) comprising the sequence shown in SEQ ID NO: 62, 63, 64, 74,75, 76, 80, 81, 82, 88, 89, 90, 93, 94, 95, 99, 100, 101, 109, 110, 111,115, 116, 117, 121, 122, 123, 132, 133,134, 137, 138, 139, 143, 144, or145; (ii) a VH CDR2 comprising the sequence shown in SEQ ID NO: 65, 66,68, 69, 70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 96, 97, 98, 102, 103,105, 106, 112, 113, 118, 119, 124, 125, 127, 128, 130, 131, 135, 136,140, 141, 146, or 147; and iii) a VH CDR3 comprising the sequence shownin SEQ ID NO: 67, 72, 73, 79, 85, 104, 107, 108, 114, 120, 126, 129,142, or 148; and/or a VL region comprising (i) a VL CDR1 comprising thesequence shown in SEQ ID NO: 149, 154, 156, 159, 162, 165, 166, 168,169, 170, 171, 173, 174, 176, 178, 181, 182, 185, 187, 190, 192, 195, or198; (ii) a VL CDR2 comprising the sequence shown in SEQ ID NO: 150,152, 155, 157, 160, 163, 172, 175, 179, 183, 186, 188, 191, 193, 196, or199; and (iii) a VL CDR3 comprising the sequence shown in SEQ ID NO:151, 153, 158, 161, 164, 167, 177, 180, 184, 189, 194, 197, or 200. Insome embodiments, the VH and VL are linked together by a flexiblelinker. In some embodiments a flexible linker comprises the amino acidsequence shown in SEQ ID NO: 202.

In some embodiments, the extracellular ligand-binding domain comprises(a) a VH region comprising (i) a VH complementarity determining regionone (CDR1) comprising the sequence shown in SEQ ID NO: 62, 63, 64, 74,75, 76, 80, 81, 82, 88, 89, 90, 109, 110, 111, 115, 116, 117, 121, 122,123, 137, 138, or 139; (ii) a VH CDR2 comprising the sequence shown inSEQ ID NO: 70, 71, 77, 78, 83, 84, 86, 87, 91, 92, 112, 113, 118, 119,124, 125, 127, 128, 140, or 141; and iii) a VH CDR3 comprising thesequence shown in SEQ ID NO: 73, 79, 85, 114, 120, 126, 129, or 142,and/or (b) a VL region comprising (i) a VL CDR1 comprising the sequenceshown in SEQ ID NO: 149, 156, 159, 162, 165, 182, 185, 187, or 195; (ii)a VL CDR2 comprising the sequence shown in SEQ ID NO: 152, 157, 160,163, 183, 186, 188, or 196; and (iii) a VL CDR3 comprising the sequenceshown in SEQ ID NO: 153, 158, 161, 164, 184, 189, or 197.

In another aspect, provided is CAR, which specifically binds toEGFRvIII, wherein the CAR comprises an extracellular ligand-bindingdomain comprising: a VH region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21, 23, 25, 27, 30, 32, 34, 35, 37, 39, 41, 43, 44, 46, 48, or50; and/or a VL region comprising VL CDR1, VL CDR2, and VL CDR3 of theVL sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 29, 31, 33, 36, 38, 40, 42, 45, 47, 49, or 51. In someembodiments, the VH and VL are linked together by a flexible linker. Insome embodiments a flexible linker comprises the amino acid sequenceshown in SEQ ID NO: 202.

In some embodiments, the CAR comprises an extracellular ligand-bindingdomain comprising: a VH region comprising a VH CDR1, VH CDR2, and VHCDR3 of the VH sequence shown in SEQ ID NO: 5, 9, 11, 13, 15, 37, 39,41, 43, or 48; and/or a VL region comprising VL CDR1, VL CDR2, and VLCDR3 of the VL sequence shown in SEQ ID NO: 6, 10, 12, 14, 16, 38, 40,42, or 49. In some embodiments, the VH and VL are linked together by aflexible linker. In some embodiments a flexible linker comprises theamino acid sequence shown in SEQ ID NO: 202.

In some embodiments, a CAR of the invention comprises an extracellularligand-binding domain having any one of partial light chain sequence aslisted in Table 1 and/or any one of partial heavy chain sequence aslisted in Table 1. In Table 1, the underlined sequences are CDRsequences according to Kabat and in bold according to Chothia. Thedifferent mAbs of Table 1 may also be referred to herein as differentanti-EGFRvIII antibody “clones”.

TABLE 1 mAb Light Chain Heavy Chain m62G7 DVVMTQTPLTLSVTIGQPASISC

EVQLQQSGPELVKPGASVKISCKT

WLLQRPG SGYTF

YTLHVVVKQSHVKSLEVVI QSPKRLIY

GVPDRFTG G

NQKFKGKATLTV SGSGTDFTLKISRVEAEDLGFYY DKSSSTAYMELRSLTSEDSAVYYC C

FGAGTKLELK AR

WGQGTSVTVSS (SEQ ID NO: 2) (SEQ ID NO: 1) h62G7 DVVMTQSPLSLPVTLGQPASISCQVQLVQSGAEVKKPGASVKVSCK

WFQQRP ASGYTF

YTLHWVRQAPGQGLE GQSPRRLIY

GVPDRFS WMG

QKFKGRVT GSGSGTDFTLKISRVEAEDVGVY MTRDTSTSTVYMELSSLRSEDTAV YC

FGGGTKVEIK YYCAR

WGQGTLVTVSS (SEQ ID NO: 4) (SEQ ID NO: 3) h62G7- DVVMTQSPLSLPVTLGQPASISCQVQLVQSGAEVKKPGASVKVSCK L6/EQ

WFQQRP ASGYTF

YTLHWVRQAPGQGLE GQSPRRLIY

GVPDRFS WMG

NQKFKGRVT GSGSGTDFTLKISRVEAEDVGVY MTRDTSTSTVYMELSSLRSEDTAV YC

FGGGTKVE1K YYCAR

WGQGTLVTVSS (SEQ ID NO: 6) (SEQ ID NO: 5) h62G7 DVVMTQSPLSLPVTLGQPASISCQVQLVQSGAEVKKPGASVKVSCK H14/

WFQQRP ASGYTF

YTLHWVRQAPGQGLE L1-DV GQSPRRLIY

GVPDRFS WMG

NQKFKGRVT GSGSGTDFTLKISRVEAEDVGVY MTRDTSTSTVYMELSSLRSEDTAV YC

FGGGTKVEIK YYCARGEAEGSWGQGTLVTVSS (SEQ ID NO: 8) (SEQ ID NO: 7) 42G9EVVLTQSPATLSVSPGERATLSC QVTLKESGPVLLKPTETLTLTCTVS

WYQQKSGQAP GFSL

MGVSWIRQPPGKALE RLLIY

GVPARFSGSGS WFA

KLSLRSRLTLSK GTEFTLTISSLQSEDFAVYYCQQ DTSKSQVVLTMTNMAPVDSATYY

FGPGTKVDIK (SEQ ID CAR

WGQGTLVTV NO: 10) SS (SEQ ID NO: 9) 32A10 EVVMTQSPATLSVSPGERVTLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQRPGQAP SGFSL

MGVSWIRQPPGKAL RLLLY

GLPGRFSGSGS EWLA

RRSLRSRLTLS GTENILTISSLQSEDFAIYFC

KDTSKSQVVLTMTNMDPVDTATY

FGPGSKVDIK (SEQ ID FCAR

WGQGTLVT NO: 12) VSS (SEQ ID NO: 11) 20B9 EIVMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQKFGQAPR SGFSL

MGVSWIRQPPGKAL LLIY

GIPVRFSGGGSG EWLG

STSLRGRITIS TEFTLTISSLQSEDFAIYSC

KDTSRGLVVLTLTNMDPVDTATYY

FGPGTTVDIK CAR

WGPGFLVTV (SEQ ID NO: 14) SS (SEQ ID NO: 13) 14C11EIVMTQSPATLSVSPGERATLSC QVTLKESGPVLVKPTETLTLTCTV

WYQQKPGQAP SGFSL

MGVSWIRQPPGKAL RLLIY

GVPARFSGSDS EWFA

RTSLRSRLTL GTEFSLTISSLQSEDFAVYFC

SKDTSKSQVVLTMTNMDPVDTAT

FGPGTKVEIK (SEQ ID YYCAR

WGQGILV NO: 16) TVSS (SEQ ID NO: 15) 21E1 I DMVVTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQPPGQSP SGFSL

MGVSWIRQPPGKAL RLLIY

GVPARFSGSGS EWFA

RRSLRSRLTLS GTDFTLTITSLESEDFAVYYC

KDTSKSQVVLTMTNMDPVDTATY

FGPGTKVDIK (SEQ ID YCAR

WGQGTLVT NO: 18) VSSN (SEQ ID NO: 17) 49B11 EMEVTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQQSGQAP SGFSL

MGVSWIRQPPGKAL RLLIS

GVPTRFSGSGS EWFA

RRSLRSRLTLS GTDFTLTITSLQSEDFAVYYC

KDTSKSQVVLTMTNMDPVDTATY

FGPGTKVDIK (SEQ ID YCAR

WGQGTLVT NO: 20) VSS (SEQ ID NO: 19) 46E10 EVVMTQSPPNLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYOORPGQSP SGFSL

MGVSWIROPPGKAL RLLLY

GVPGRFSGSG EWLA

RRSLRSRLTLS SGTENILTISSLQSEDFAVYFC

KDTSKSQVVLIMTNMDPVDTATYY

FGPGSKVDIK (SEQ ID CAR

WGQGTLVTV NO: 22) SS (SEQ ID NO: 21) 12H6 EVVMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQRPGQSP SGFSL

MGVSWIRQPPGKAL RLLLY

GVPGRFSGSG EWLA

RRSLRSRLTLS SGTENILTISSLQSEDFAIYFC

KDTSKSQWLTMTNMDPVDTATY

FGPGSKVDIK (SEQ ID YCAR

WGQGTLVT NO: 24) VSS (SEQ ID NO: 23) 19A9 EVVMTQSPATLSVSPGERATLSCQVTLEESGPVLVKPTETLTLTCTV

WYQQKPGQAP SGFSL

MGVSWIRQPPGKAP RLLIF

GIPARFSGSGSG EWFA

RLSLRSRLTL TEFTLTIDSLQSEHSGLYYC

SKDTSKSQVVLTMTNMDPVDTAT

FGPGTKVDIK (SEQ ID YYCAR

WGQGTLV NO: 26) TVSS (SEQ ID NO: 25) 11B11 EVLMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQRPGQAP SGFSL

MGVSWIRQPPGKAL RLLLF

GIPGRFSGSGS EWLA

RRSLRSRLTM GTENILTISSLQSEDFAIYFC

SKDTSKSQVVLTMTNMDPVDTAT

FGPGSKVEIK (SEQ ID YYCVR

WGQGTLV NO: 28) TVSS (SEQ ID NO: 27) 21E7 DVVLTQSPATLSVSPGERATLSCQVTLEESGPVLVKPTETLTLTCTV

WYQQNPGQAP SGFSL

MGVSWIRQPPGKAP RLLIF

GIPASFSGSGSG EWFA

RLSLRSRLTL TEFTLTINSLQSEHSAVYYC

SKDTSKSQVVLTMTNMDPVDTAT

FGPGTKVDIK (SEQ ID YYCAR

WGQGTLV NO: 29) TVSS (SEQ ID NO: 25) 12B2 EVVMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQKPGQAPR SGFSL

MGVSWIRQPPGKAL LLIY

DIPARFSGSGSGT EWLG

RLSLRSRLSIS EFTLTISSLQSEDFAVYYC

KDTSKSQVVLTMTNMDPVDTATY

FGPGTKVDIK (SEQ ID YCVR

WGQGTLV NO: 31) TVSS (SEQ ID NO: 30) 11F10 EIVMTQSPATLSVSPGERTTLSCQVTLKESGPVLVKPIETLTLTCTVC

WYQQKPGQAP GFSL

MGVSWIRQPPGKALE RLLIY

VPARFSGSGS WLG

RLSISK GTEFTLTISSLQSEDFAVYSC

DTSKSQVVLTMTNMDPVDTATYY

FGQGTIWEIK (SEQ ID CAR

WGQGTLVTV NO: 33) SS (SEQ ID NO: 32) 17G11 EVVMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTVF

WYQQKPGQAPR GFSL

MGVSWIRQPPGKAPE LLIY

DIPARFSGSGSGT WLG

RLSLRSRLSISK EFTLTISSLQSEDFAVYYC

DTSKSQVVFXMTNMDPGDPATYY

FGPGTKVDIK (SEQ ID CVR

WGQGTLVTV NO: 31) SS (SEQ ID NO: 34) 29D5 KIVMTQSPATLSVSPGERATLSCQVTLKESGPVLVKPTETLTLTCTV

WYQQKPGQAPR SGFSL

MGVSWLRQPPGKAL LLVF

GIPIRFSGSGSGT EWFA

SPSLRGRLTV EFTLTVSSLQSEDFAVYVC

SKDTSKSQWLTLTNMDPVDTATY

FGPGTKVDIK (SEQ ID YCAR

WGQGTLVT NO: 36) VSS (SEQ ID NO: 35) 30D8 DIVMTQSPLSLPVTPGEPASISC

EVQLVESGGGLVKPGGSLRLSCE

WFLQKPG ASGFTF

AWMSWVRQAPGKGL QSPQLLIY

GVPDRFSG EWVG

VVPLNG GGSGTDFTLKISRVEAEDVGVYY RFIISRDDSRNTLYLQLNNLKTEDT C

FGQGTRLEIK AVYYCTT

WGQGTLVTV (SEQ ID NO: 38) SS (SEQ ID NO: 37) 20E12DIVLTQSPLSLSVTPGEPASISC

R EVNLVESGGGLVKPGGSLRLSCE

WFLHKPG ASGFTF

AWMSWVRQAPGKGL OSPOLLIY

GVPDRFSG EWVG

AAPVRN SGSGIDFILKISRVEAEDVGVYYC RFTISRDDSRNTLYLEMHSLKTED

FGQGTRLEIK (SEQ TAVYYCTT

WGQGTM ID NO: 40) VTVSS (SEQ ID NO: 39) 26B9 DIVLTQSPLSLPVTPGEPASISC

EVQLVESWGVLVKPGGSLRLSCA

WFLQKPG ASGFIF

AWMSVWRQAPGKGLE QSPQLLIY

GVPDRFSG WIG

RF SDSGTDFTLKISRVEAEDVGVYY TISRDDSKDTLYLMINGLKTEDTA C

FGQGTRLEIK VYFCTT

WGQGTLVTV (SEQ ID NO: 42) SS (SEQ ID NO: 41) 32G8DIVLTQSPLSLSVTPGEPASISC

EVNLVESGGGLVKPGGSLRLSCE

WFLHKPG ASGFTF

AWMSWVRQAPGKGL QSPQLLIY

GVPDRFSG EVWG

AAPVRNR SGSGIDFILKISRVEAEDVGVYYC CTISRDDSRNTLYLEMHSLKTEDT

FGQGTRLEIK (SEQ AVYYCTT

WGQGRM ID NO: 40) VTVSS (SEQ ID NO: 43) 34E7 DIVLTQSPLSLSVTPGEPASISC

EVNLVESGGGLVKPGGSLRLSCE

WFLHKPG ASGFTF

AWMSWVRQAPGKGL QSPQLLIF

GVPDRFSG EVWG

ASPVRN SGSGIDFILKISRVEAEDVGVYYC RFTISRDDSRNMLYLEMHSLKTED

FGQGTRLEIK (SEQ TAVYYCTT

WGQGTL ID NO: 45) VTVSS (SEQ ID NO: 44) 20G5 DIVLTQSPLSLPVTPGEPASISC

EVQLVESGGDLVKPGGSLRLSCA

WFLOKPG ASGFTF

AWMSWVRQAPGKGL QSPHLLIY

GVPDRFSG EVWG

AAPVKG SGSGTDFTLKISRVEAEDVGVYY RFIISRDDSKNILSLOMNSLKTEDT C

FGQGTRLEIK (SEQ AMYYCTT

WGQGSLV ID NO: 47) TVSS (SEQ ID NO: 46) C6 ELQSVLTQPPSASGTPGQRVTISQVQLVQSGAEVKKPGSSVKVSCK C

WYQQLPGT ASGDTF

NAISWVRQAPGQGLE APKILIY

GVPDRFSGS WMG

AQKFQGRVTIT KSGTSASLAISGLRSEDEADYYC ADESTSTAYMELSSLRSEDTAVYY

FGTGTKLTVL CAR

WG (SEQ ID NO: 49) QGTLVTVSS (SEQ ID NO: 48) B5 DIQMTQSPSSLSASVGDRVTITCEVQLLESGGGLVQPGGSLRLSCA

WYQQKPGKAPK ASGFTF

YAMSWVRQAPGKGLE LLIY

GVPSRFSGSGSG WVS

ADSVKGRFTI TDFTLTISSLQPEDFATYYC

SRDNSKNTLYLQMNSLRAEDTAV

FGQGTKVEIK (SEQ ID YYCAR

WGQ NO: 51) GTLVTVSS (SEQ ID NO: 50)

Also provided herein are CDR portions of extracellular ligand-bindingdomains of CARs to EGFRvIII (including Chothia, Kabat CDRs, and CDRcontact regions). Determination of CDR regions is well within the skillof the art. It is understood that in some embodiments, CDRs can be acombination of the Kabat and Chothia CDR (also termed “combined CRs” or“extended CDRs”). In some embodiments, the CDRs are the Kabat CDRs. Inother embodiments, the CDRs are the Chothia CDRs. In other words, inembodiments with more than one CDR, the CDRs may be any of Kabat,Chothia, combination CDRs, or combinations thereof. Table 2 providesexamples of CDR sequences provided herein.

TABLE 2 Heavy Chain mAb CDRH1 CDRH2 CDRH3 m62G7 TDYTLH (SEQ ID NO: 62)GIDPINGGITINQKEK GEAMDS (SEQ ID (Kabat); G (SEQ ID NO: 65) NO: 67)GYTFTD (SEQ ID NO: 63) (Kabat) (Chothia); GIDPINGGTTY (SEQGYTFTDYTLH (SEQ ID NO: ID NO: 66)(Chothia) 64)(extended) h62G7TDYTLH (SEQ ID NO: 62) GINPINGGTTYNQKFIK GEAMDS (SEQ ID (Kabat);G (SEQ ID NO: 68) NO: 67) GYTFTD (SEQ ID NO: 63) (Kabat) (Chothia);GINPINGGTTY (SEQ GYTFTDYTLH (SEQ ID NO: ID NO: 69)(Chothia)64)(extended) h62G7-H14 TDYTLH (SEQ ID NO: 62) GIWPITGGTTYNQKFKGEAEGS (SEQ ID (Kabat); G (SEQ ID NO: 70) NO: 72) GYTFTD (SEQ ID NO: 63)(Kabat) (Chothia); GIWPITGGTTY (SEQ GYTFTDYTLH (SEQ ID NO:ID NO: 71)(Chothia) 64)(extended) h62G7-EQ TDYTLH (SEQ ID NO: 62)GIWPITGGTTYNQKFK GEAQGS (SEQ ID (Kabat); G (SEQ ID NO: 70) NO: 73)GYTFTD (SEQ ID NO: 63) (Kabat) (Chothia); GIWPITGGTTY (SEQGYTFTDYTLH (SEQ ID NO: ID NO: 71)(Chothia) 64)(extended) 42G9SNPRMGVS (SEQ ID NO: 74) HIFSTDEKSLKLSLRS DSSNYEGYFDF (Kabat);(SEQ ID NO: 77)(Kabat) (SEQ ID NO: 79) GFSLSNPR (SEQ ID NO: 75)HIFSTDEKSL (SEQ ID (Chothia); NO: 78)(Chothia) GFSLSNPRMGVS (SEQ IDNO: 76)(extended) 32A10 SNARMGVS (SEQ ID NO: 80) HIFSTDEKSIRRSLRSDSSNYEGYFDY (Kabat); (SEQ ID NO: 83) (SEQ ID NO: 85)GFSLSNAR (SEQ ID NO: 81) (Kabat) (Chothia); HIESIDEKSI (SEQ IDGFSLSNARMGVS (SEQ ID NO: 84)(Chothia) NO: 82)(extended) 20B9SNARMGVS (SEQ ID NO: 80) HIFSTDEKSYSTSLRG DSSNYEGYFDF (Kabat);(SEQ ID NO: 86)(Kabat) (SEQ ID NO: 79) GFSLSNAR (SEQ ID NO: 81)HIFSTDEKSY (SEQ ID (Chothia); NO: 87)(Chothia) GFSLSNARMGVS (SEQ IDNO: 82)(extended) 14C11 NNARMGVS (SEQ ID NO: HIFSTDEKSFRTSLRSDSSNYEGYFDY 88)(Kabat); (SEQ ID NO: 91)(Kabat) (SEQ ID NO: 85)GFSLNNAR (SEQ ID NO: 89) HIFSTDEKSF (SEQ ID (Chothia); NO: 92)(Chothia)GFSLNNARMGVS (SEQ ID NO: 90)(extended) 21E11 SNVRMGVS (SEQ ID NO: 93)HIFSSDEKSIRRSLRSE DSSNYEGYFDF (Kabat); (SEQ ID NO: 96)(Kabat)(SEQ ID NO: 79) GFSLSNVR (SEQ ID NO: 94) HIFSSDEKSI (SEQ ID (Chothia);NO: 97)(Chothia) GFSLSNVRMGVS (SEQ ID NO: 95)(extended) 49B11SNVRMGVS (SEQ ID NO: 93) HIFSSDEKSIRRSLRS DSSNYEGYFDY (Kabat);(SEQ ID NO: 96)(Kabat) (SEQ ID NO: 85) GFSLSNVR (SEQ ID NO: 94)HIFSSDEKSI (SEQ ID (Chothia); NO: 97)(Chothia) GFSLSNVRMGVS (SEQ IDNO: 95)(extended) 46E10 SNARMGVS (SEQ ID NO: 80) HIFSTDEKSIRRSLRSDSSNYEGYFDY 12H6 (Kabat); (SEQ ID NO: 83) (SEQ ID NO: 85)GFSLSNAR (SEQ ID NO: 81) (Kabat) (Chothia); HIFSTDEKSI (SEQ IDGFSLSNARMGVS (SEQ ID NO: 84)(Chothia) NO: 82)(extended) 19A9SNARMGVS (SEQ ID NO: 80) HIFSTDEKSLRLSLRS DSSNYEGYFDY 21E7 (Kabat);(SEQ ID NO: 98) (SEQ ID NO: 85) GFSLSNAR (SEQ ID NO: 81) (Kabat)(Chothia); HIFSTDEKSL (SEQ ID GFSLSNARMGVS (SEQ ID NO: 78)(Chothia)NO: 82)(extended) 11B11 SNAKMGVS (SEQ ID NO: 99) HIFSTDEKSIRRSLRSDSSNYEGYFDY (Kabat); (SEQ ID NO: 83) (SEQ ID NO: 85)GFSLSNAK (SEQ ID NO: 100) (Kabat) (Chothia); HIFSTDEKSI (SEQ IDGFSLSNAKMGVS (SEQ ID NO: 84)(Chothia) NO: 101)(extended) 12B2SNPRMGVS (SEQ ID NO: 74) HIFSSDEKSYRLSLRS DSSNYGGYFDY (Kabat);(SEQ ID NO: 102) (SEQ ID NO: 104) GFSLSNPR (SEQ ID NO: 75) (Kabat)(Chothia); HIFSSDEKSY (SEQ ID GFSLSNPRMGVS (SEQ ID NO: 103)(Chothia)NO: 76)(extended) 11F10 SNPRMGVS (SEQ ID NO: 74) HIFSSDEKSYRLFLRSDSSDYEGYFDY (Kabat); (SEQ ID NO: 105) (SEQ ID NO: 107)GFSLSNPR (SEQ ID NO: 75) (Kabat) (Chothia); HIFSSDEKSY (SEQ IDGFSLSNPRMGVS (SEQ ID NO: 103)(Chothia) NO: 76)(extended) 17G11SNPRMGVS (SEQ ID NO: 74) HIFSSDEKSYRLSLRS DSSNYEEYFDY (Kabat);(SEQ ID NO: 102) (SEQ ID NO: 108) GFSLSNPR (SEQ ID NO: 75) (Kabat)(Chothia); HIFSSDEKSY (SEQ ID GFSLSNPRMGVS (SEQ ID NO: 103)(Chothia)NO: 76)(extended) 29D5 SNPRMGVS (SEQ ID NO: 74) HIFSTDEKSYSPSLRGDSSNYEGYFDY (Kabat); (SEQ ID NO: 106) (SEQ ID NO: 85)GFSLSNPR (SEQ ID NO: 75) (Kabat) (Chothia); HIFSTDEKSY (SEQ IDGFSLSNPRMGVS (SEQ ID NO: 87)(Chothia) NO: 76)(extended) 30D8SDAWMS (SEQ ID NO: 109) RIKSKTDGGTTDYVVPL VPGSYGY (SEQ ID (Kabat); NGNO: 114) GFTFSD (SEQ ID NO: 110) (SEQ ID NO: 112) (Chothia); (Kabat)GFTFSDAWMS (SEQ ID NO: RIKSKTDGGTTDY 111)(extended) (SEQ ID NO: 113)(Chothia) 20E12 SYAWMS (SEQ ID NO: 115) RIKSIADGGATDYAAP IPGNDAFDM (SEQ(Kabat); VRN (SEQ ID NO: ID NO: 120) GFTFSY (SEQ ID NO: 116) 118)(Kabat)(Chothia); RIKSIADGGATDY (SEQ GFTFSYAWMS (SEQ ID NO:ID NO: 119)(Chothia) 117)(extended) 26B9 NNAWMS (SEQ ID NO: 121)RIKSKSDGGTTDYAAP APGGPFDY (SEQ (Kabat); VKD (SEQ ID NO: 124) ID NO: 126)GFIFNN (SEQ ID NO: 122) (Kabat) (Chothia); RIKSKSDGGTTDYGFIFNNAWMS (SEQ ID NO: (SEQ ID NO: 125) 123)(extended) (Chothia) 32G8SYAWMS (SEQ ID NO: 115) RIKSITDGGVIDYAAPV IPGNDDFDM (Kabat);RN (SEQ ID NO: 127) (SEQ ID NO: 129) GFTFSY (SEQ ID NO: 116) (Kabat)(Chothia); RIKSITDGGVIDY (SEQ GFTFSYAWMS (SEQ ID NO:ID NO: 128)(Chothia) 117)(extended) 34E7 SYAWMS (SEQ ID NO: 115)RIKSINDGGATDYASPV IPGNDAFDM (SEQ (Kabat); RN (SEQ ID NO: 130)ID NO: 120) GFTFSY (SEQ ID NO: 116) (Kabat) (Chothia); RIKSINDGGATDYGFTFSYAWMS (SEQ ID NO: (SEQ ID NO: 131 117)(extended) (Chothia) 20G5TNAWMS (SEQ ID NO: 132) RIKSKIDGGTTDYAAPV APGGPFDY (SEQ (Kabat);KG (SEQ ID NO: 135) ID NO: 126) GFTFTN (SEQ ID NO: 133) (Kabat)(Chothia); RIKSKIDGGTTDY (SEQ GFTFTNAWMS (SEQ ID NO:ID NO: 136)(Chothia) 134)(extended) C6 SSNAIS (SEQ ID NO: 137)VIIPIFGTADYAQKFQG HTYHEYAGGYYGG (Kabat); (SEQ ID NO: 140) AMDP (SEQ IDGDTFSS (SEQ ID NO: 138) (Kabat) NO: 142) (Chothia); VIIPIFGTADY (SEQ IDGDTFSSNAIS (SEQ ID NO: NO: 141)(Chothia) 139)(extended) B5SNYAMS (SEQ ID NO: 143) DISGGGGRTYYADSVK AGLLYGGGVYPM (Kabat);G (SEQ ID NO: 146) DI (SEQ ID NO: GFTFSN (SEQ ID NO: 144) (Kabat) 148)(Chothia); DISGGGGRTYY (SEQ GFTFSNYAMS (SEQ ID NO: ID NO: 147)(Chothia)145)(extended) Light Chain mAb CDRL1 CDRL2 CDRL3 m62G7KSSQSLLYSNGKTYLN (SEQ ID LVSKLDS (SEQ ID NO: VQDTHFPLT (SEQ h62G7NO: 149) 150) ID NO: 151) h62G7-L6 KSSQSLLYSNGKTYLN (SEQ IDQVSKLDS (SEQ ID NO: GQDTHFPLT (SEQ NO: 149) 152) ID NO: 153) h62G7-L1-DVKSSQSLLYSNDKTYTN (SEQ EVSKLDV (SEQ ID NO: GQDTHFPLT (SEQ ID NO: 154)155) ID NO: 153) 42G9 RASQSVRSNLA (SEQ ID NO: GSTIRAT (SEQ ID NO:QQYSDWPFT 156) 157) (SEQ ID NO: 158) 32A10 RASQSVSSNFA (SEQ ID NO:GATTRAT (SEQ ID NO: QQYKDWPFT 159) 160) (SEQ ID NO: 161) 20B9RVSQSIGANLA (SEQ ID NO: GASTRAT (SEQ ID NO: QQYIYWPFT (SEQ 162) 163)ID NO: 164) 14C11 RASQSVSNNLA (SEQ ID NO: GASTRAT (SEQ ID NO: QQYKDWPFT165) 163) (SEQ ID NO: 161) 21E11 RASQSVGSDLA (SEQ ID NO:GASTRAT (SEQ ID NO: QQYNDWPFT 166) 163) (SEQ ID NO: 167) 49B11RASQNIGSDLA (SEQ ID NO: GASTRAT (SEQ ID NO: QQYNDWPFT 168) 163)(SEQ ID NO: 167) 46E10 RASQSVTSNFA (SEQ ID NO: GASTRAT (SEQ ID NO:QQYKDWPFT 169) 163) (SEQ ID NO: 161) 12H6 RASQGVSSNFA (SEQ ID NO:GASTRAT (SEQ ID NO: QQYKDWPFT 170) 163) (SEQ ID NO: 161) 19A9RASQSVNRNLA (SEQ. ID NO: GTSTRAT (SEQ ID NO: QQYNDWPFT 171) 172)(SEQ ID NO: 167) 11B11 RASQSVSTNFA (SEQ ID NO: GASTRAT (SEQ ID NO:QQYKDWPFT 173) 163) (SEQ ID NO: 161) 21E7 RASQSVNSNLA (SEQ ID NO:GSSTRAT (SEQ ID NO: QQYNDWPFT 174) 175) (SEQ ID NO: 167) 12B2RASQSVINNLA (SEQ ID NO: GTSTRAT (SEQ ID NO: QDYNNWPFT 17G11 176) 172)(SEQ ID NO: 177) 11E10 RASQSVGSNLA (SEQ ID NO: GASTRASG (SEQ IDQEYNNWPFT 178) NO: 179) (SEQ ID NO: 180) 29D5 RANQIVSSNLA (SEQ ID NO:GTSTRAT (SEQ ID NO: QQYNDWPFT 181) 172) (SEQ ID NO: 167) 30D8RSSQSLLHNKRNNYLD (SEQ LASNRAS (SEQ ID NO: MQAQQTPIT (SEQ ID NO: 182)183) ID NO: 184) 20E12 RSSQSLLYSNGKNYLD (SEQ LGSNRAS (SEQ ID NO:MQAQQTPIT (SEQ 32G8 ID NO: 185) 186) ID NO: 184) 26B9RSSQSLLHRDGFNYLD (SEQ LASSRAS (SEQ ID NO: MQALQTPIT (SEQ ID NO: 187)188) ID NO: 189) 34E7 RSTQSLLYSNGKNYLD (SEQ LGSIRAS (SEQ ID NO:MQAQQTPIT (SEQ ID NO: 190) 191) ID NO: 184) 20G5 RSSQSLLYSDRRNYLD (SEQLGSYRAS (SEQ ID NO: MQALQIPIT(SEQ ID NO: 192) 193) ID NO: 194) C6SGSSSNIGSNYVY RNNQRPS AAWDDNLSGWV (SEQ ID NO: 195) (SEQ ID NO: 196)(SEQ ID NO: 197) B5 RASQSISSYLN AASSLQS (SEQ ID NO: QQSYSTPLT(SEQ(SEQ ID NO: 198) 199) ID NO: 200)

The invention encompasses modifications to the CARs and polypeptides ofthe invention variants shown in Table 1, including functionallyequivalent CARs having modifications which do not significantly affecttheir properties and variants which have enhanced or decreased activityand/or affinity. For example, the amino acid sequence may be mutated toobtain an antibody with the desired binding affinity to EGFRvIII.Modification of polypeptides is routine practice in the art and need notbe described in detail herein. Examples of modified polypeptides includepolypeptides with conservative substitutions of amino acid residues, oneor more deletions or additions of amino acids which do not significantlydeleteriously change the functional activity, or which mature (enhance)the affinity of the polypeptide for its ligand, or use of chemicalanalogs.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto an epitope tag. Other insertional variants of the antibody moleculeinclude the fusion to the N- or C-terminus of the antibody of an enzymeor a polypeptide which increases the half-life of the antibody in theblood circulation.

Substitution variants have at least one amino acid residue in theantibody molecule removed and a different residue inserted in its place.The sites of greatest interest for substitutional mutagenesis includethe hypervariable regions, but FR alterations are also contemplated.Conservative substitutions are shown in Table 3 under the heading of“conservative substitutions.” If such substitutions result in a changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 3, or as further described below inreference to amino acid classes, may be introduced and the productsscreened. In some embodiments, substitution variants of antibodiesprovided herein have no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, 2, or conservative substitution in the VH or VL region as comparedto the reference parent antibody. In some embodiments, the substitutionsare not within a CDR of the VH or VL region.

TABLE 3 Amino Acid Substitutions Original Residue (naturally occurringConservative Exemplary amino acid) Substitutions Substitutions Ala (A)Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp.Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; GluGu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)Leu Ile; Leu; Met; Phe; Ala; Norleucine

In some embodiments, the invention provides a CAR comprising anextracellular ligand-binding domain that binds to EGFRvIII and competesfor binding to EGFRvIII with the antibodies described herein or the CARsdescribed herein (e.g., Table 5A), including m62G7, h62G7,h62G7-H14/L1-DV, h62G7-L6/EQ, 42G9, 32A10, 20B9, 14C11, 21E11, 49B11,46E10, 12H6, 19A9, 21E7, 11B11, 12B2, 11F10, 17G11, 29D5, 30D8, 20E12,26B9, 32G8, 34E7, 20G5, C6, and B5.

In some embodiments, the invention provides CARs comprising CDR portionsof antibodies to EGFRvIII antibodies based on CDR contact regions. CDRcontact regions are regions of an antibody that imbue specificity to theantibody for an antigen. In general, CDR contact regions include theresidue positions in the CDRs and Vernier zones which are constrained inorder to maintain proper loop structure for the antibody to bind aspecific antigen. See, e.g., Makabe et al., J. Biol. Chem.,283:1156-1166, 2007. Determination of CDR contact regions is well withinthe skill of the art.

The binding affinity (K_(D)) of the EGFRvIII specific CAR as describedherein to EGFRvIII (such as human EGFRvIII (e.g., (SEQ ID NO: 201)) canbe about 0.001 to about 5000 nM. In some embodiments, the bindingaffinity is about any of 5000 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM,2500 nM, 2000 nM, 1789 nM, 1583 nM, 1540 nM, 1500 nM, 1490 nM, 1064 nM,1000 nM, 933 nM, 894 nM, 750 nM, 705 nM, 678 nM, 532 nM, 500 nM, 494 nM,400 nM, 349 nM, 340 nM, 353 nM, 300 nM, 250 nM, 244 nM, 231 nM, 225 nM,207 nM, 200 nM, 186 nM, 172 nM, 136 nM, 113 nM, 104 nM, 101 nM, 100 nM,90 nM, 83 nM, 79 nM, 74 nM, 54 nM, 50 nM, 45 nM, 42 nM, 40 nM, 35 nM, 32nM, 30 nM, 25 nM, 24 nM, 22 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15nM, 12 nM, 10 nM, 9 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM,4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, or 0.001 nM. Insome embodiments, the binding affinity is less than about any of 5000nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 250 nM, 200 nM,100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM,4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, or 0.5 nM.

The intracellular signaling domain of a CAR according to the inventionis responsible for intracellular signaling following the binding ofextracellular ligand-binding domain to the target resulting in theactivation of the immune cell and immune response. The intracellularsignaling domain has the ability to activate of at least one of thenormal effector functions of the immune cell in which the CAR isexpressed. For example, the effector function of a T cell can be acytolytic activity or helper activity including the secretion ofcytokines.

In some embodiments, an intracellular signaling domain for use in a CARcan be the cytoplasmic sequences of, for example without limitation, theT cell receptor and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability. Intracellular signaling domainscomprise two distinct classes of cytoplasmic signaling sequences: thosethat initiate antigen-dependent primary activation, and those that actin an antigen-independent manner to provide a secondary orco-stimulatory signal. Primary cytoplasmic signaling sequences cancomprise signaling motifs which are known as immunoreceptortyrosine-based activation motifs of ITAMs. ITAMs are well definedsignaling motifs found in the intracytoplasmic tail of a variety ofreceptors that serve as binding sites for syk/zap70 class tyrosinekinases. Examples of ITAM used in the invention can include as nonlimiting examples those derived from TCRζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ,CD3ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, theintracellular signaling domain of the CAR can comprise the CD3ζ (zeta)signaling domain which has amino acid sequence with at least about 70%,preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99%sequence identity with an amino acid sequence shown in SEQ. ID NO: 205.In some embodiments the intracellular signaling domain of the CAR of theinvention comprises a domain of a co-stimulatory molecule.

In some embodiments, the intracellular signaling domain of a CAR of theinvention comprises a part of co-stimulatory molecule selected from thegroup consisting of a fragment of 41BB (GenBank: AAA53133.) and CD28(NP_006130.1). In some embodiments, the intracellular signaling domainof the CAR of the invention comprises amino acid sequence whichcomprises at least 70%, preferably at least 80%, more preferably atleast 90%, 95%, 97%, or 99% sequence identity with an amino acidsequence shown in SEQ. ID NO: 213 (CD28 signaling domain) or SEQ. ID NO:204 (4-1BB signaling domain).

CARs are expressed on the surface membrane of the cell. Thus, the CARcan comprise a transmembrane domain. Suitable transmembrane domains fora CAR disclosed herein have the ability to (a) be expressed at thesurface of a cell, preferably an immune cell such as, for examplewithout limitation, lymphocyte cells or Natural killer (NK) cells, and(b) interact with the ligand-binding domain and intracellular signalingdomain for directing cellular response of immune cell against apredefined target cell. The transmembrane domain can be derived eitherfrom a natural or from a synthetic source. The transmembrane domain canbe derived from any membrane-bound or transmembrane protein. Asnon-limiting examples, the transmembrane polypeptide can be α subunit ofthe T cell receptor such as α, β, γ or δ, polypeptide constituting CD3complex, IL-2 receptor p55 (a chain), p75 (β chain) or γ chain, subunitchain of Fc receptors, in particular Fcγ receptor III or CD proteins.Alternatively, the transmembrane domain can be synthetic and cancomprise predominantly hydrophobic residues such as leucine and valine.In some embodiments said transmembrane domain is derived from the humanCD8α chain (e.g., NP_001139345.1). The CAR can further comprise a stalkdomain between the extracellular ligand-binding domain and saidtransmembrane domain. A stalk domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. Stalk region may be derived from all or part of naturallyoccurring molecules, such as from all or part of the extracellularregion of CD8, CD4, or CD28, or from all or part of an antibody constantregion. Alternatively the stalk domain may be a synthetic sequence thatcorresponds to a naturally occurring stalk sequence, or may be anentirely synthetic stalk sequence. In some embodiments said stalk domainis a part of human CD8α chain (e.g., NP_001139345.1). In anotherparticular embodiment, said transmembrane and hinge domains comprise apart of human CD8α chain, preferably which comprise at least 70%,preferably at least 80%, more preferably at least 90%, 95%, 97%, or 99%sequence identity with the amino acid sequence shown in SEQ ID NO: 210and SEQ ID NO: 208, respectively. In some embodiments, CARs disclosedherein can comprise an extracellular ligand-binding domain thatspecifically binds EGFRvIII, CD8a human hinge and transmembrane domains,the CD34 signaling domain, and the 4-1BB signaling domain.

Table 4 provides exemplary sequences of domains which can be used in theCARs disclosed herein.

TABLE 4 Exemplary sequences of CAR Components SEQ ID DomainAmino Acid Sequence NO: CD8α signal peptide MALPVTALLLPLALLLHAARP 206FcγRIIIα hinge GLAVSTISSFFPPGYQ 207 CD8α hingeTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 208 IgG1 hingeEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDV 209SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK CD8α IYIWAPLAGTCGVLLLSLVITLYC 210transmembrane (TM) domain 41BB intracellularKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 203 signaling domain (ISD)CD3ζ intracellular RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 205signaling domain KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST (ISD)ATKDTYDALHMQALPPR FcϵRI α-TM-IC (FcϵRIFFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRKGFRLLNPHPKPNPKNN 211 α chaintransmembrane and intracellular domain) FcϵRIP-ΔITAM (FcϵRIMDTESNRRANLALPQEPSSVPAFEVLEISPQEVSSGRLLKSASSPPLHTW 212 β chain withoutLTVLKKEQEFLGVTQILTAMICLCFGTVVCSVLDISHIEGDIFSSFKAGYPF ITAM)WGAIFFSISGMLSIISERRNATYLVRGSLGANTASSIAGGTGITILIINLKKSLAYIHIFISCQKFFETKCFMASFSTEIVVMMLFLTILGLGSAVSLTICGAGEEL KGNKVPE41BB-IC (41BB co- KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 204signaling domain) CD28-IC (CD28 co-RSKRSRGGFISDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 213 signaling domainFcϵRlγ-SP (signal MIPAVVLLLLLLVEQAAA 214 peptide) FcϵRl γ-ΔITAMLGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKS 215 (FcϵERl γ chainwithout ITAM) GSG-P2A (GSG-P2A GSGATNFSLLKQAGDVEENPGP 216 ribosomal skippolypeptide) GSG-T2A (GSG-T2A GSGEGRGSLLTCGDVEENPGP 217 ribosomal skippolypeptide)

Table 5A provides amino acid sequences of exemplary EGFRvIII specificCARs of the present invention. In Table 5A, the signal/leader peptidesequence is in bold, and GS linker [(GGGGS)₄ (SEQ ID NO: 202)] isunderlined.

TABLE 5A Amino acid sequences of exemplary EGFRvIII specific CARsComponents in order, N-terminus CAR CAR Amino Acid Sequenceto C-terminus) h62G7- MALPVTALLLPLALLLHAARPDVVMTQSPLSLPVTLGQPCD8α signal peptide; L6/EQ ASISCKSSQSLLYSNGKTYLNWFQQRPGQSPRRLIYQVSh62G7-L6/EQVL (Table KLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQD1 SEQ ID NO: 6); THFPLTFGGGTKVEIKGGGGSGGGGSGGGGSGGGGSQVQGS linker [(GGGGS)₄ LVQSGAEVKKPGASVKVSCKASGYTFTDYTLHWVRQAPG(SEQ ID NO: 202)]; QGLEWMGGIWPITGGTTYNQKFKGRVTMTRDTSTSTVYM h62G7-L6/EQVHELSSLRSEDTAVYYCARGEAQGSWGQGTLVTVSSTTTPA (Table 1 SEQ ID NO: 5);PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFAC CD8α hinge;DIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP CD8α TM;FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP 4-1BB signalingAYQQGQKQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR domain;RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGL CD3zeta signalingYQGLSTATKDTYDALHMQALPPR domain (SEQ ID NO: 52) 42G9MALPVTALLLPLALLLHAARPQVTLKESGPVLLKPTETL CD8α signal peptide;TLTCTVSGFSLSNPRMGVSWIRQPPGKALEWFAHIFSTD 42G9 VH (Table 1 SEQEKSLKLSLRSRLTLSKDTSKSQVVLTMTNMAPVDSATYY ID NO: 9);CARDSSNYEGYFDFWGQGTLVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEVVLTQSPATLSVSPGERATLSCRASQSVRSNL 42G9 VL (Table 1 SEQAWYQQKSGQAPRLLIYGSTIRATGVPARFSGSGSGTEFT ID NO: 10);LTISSLQSEDFAVYYCQQYSDWPFTFGPGTKVDIKTTTP CD8α hinge;APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α TM;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ 4-1BB signalingPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA domain;PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3zeta signalingRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG domain LYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 53) 32A10 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLCD8α signal peptide; TLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSTD32A10 VH (Table 1 SEQ EKSIRRSLRSRLTLSKDTSKSQVVLTMTNMDPVDTATYFID NO: 11); CARDSSNYEGYFDYWGQGTLVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEVVMTQSPATLSVSPGERVTLSCRASQSVSSNF 32A10 VL (Table 1 SEQAWYQQRPGQAPRLLLYGATTRATGLPGRFSGSGSGTENI ID NO: 12);LTISSLQSEDFAIYFCQQYKDWPFTFGPGSKVDIKTTTP CD8α hinge;APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α TM;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ 4-1BB signalingPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA domain;PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3zeta signalingRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG domainLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 54) 20B9MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETL CD8α signal peptide;TLTCTVSGFSLSNARMGVSWIRQPPGKALEWLGHIFSTD 20B9 VH (Table 1, SEQEKSYSTSLRGRITISKDTSRGLVVLTLTNMDPVDTATYY ID NO: 13);CARDSSNYEGYFDFWGPGFLVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEIVMTQSPATLSVSPGERATLSCRVSQSIGANL 20B9 VL (Table 1, SEQAWYQQKFGQAPRLLIYGASTRATGIPVRFSGGGSGTEFT ID NO: 14);LTISSLQSEDFAIYSCQQYIYWPFTFGPGTTVDIKTTTP CD8α hinge;APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α TM;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ 4-1BB signalingPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA domain;PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3zeta signalingRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG domain LYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 55) 14C11 MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLCD8α signal peptide; TLTCTVSGFSLNNARMGVSWIRQPPGKALEWFAHIFSTD14C11 VH (Table 1, SEQ EKSFRTSLRSRLTLSKDTSKSQVVLTMTNMDPVDTATYYID NO: 15); CARDSSNYEGYFDYWGQGILVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSNNL 14C11 VL (Table 1, SEQAWYQQKPGQAPRLLIYGASTRATGVPARFSGSDSGTEFS ID NO: 16);LTISSLQSEDFAVYFCQQYKDWPFTFGPGTKVEIKTTTP CD8α hinge;APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α TM;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ 4-1BB signalingPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA domain;PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3zeta signalingRRKNPQEGLYKELQKDKMAEAYSEIGMKGERRRGKGHDG domain LYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 56) 20E12 MALPVTALLLPLALLLHAARPEVNLVESGGGLVKPGGSLCD8α signal peptide; RLSCEASGFTFSYAWMSWVRQAPGKGLEWVGRIKSIADG20E12 VH (Table 1, SEQ GATDYAAPVRNRFTISRDDSRNTLYLEMHSLKTEDTAVYID NO: 39); YCTTIPGNDAFDMWGQGTMVTVSSGGGGSGGGGSGGGGS GS linker;GGGGSDIVLTQSPLSLSVTPGEPASISCRSSQSLLYSNG 20E12 VL (Table 1, SEQKNYLDWFLHKPGQSPQLLIYLGSNRASGVPDRFSGSGSG ID NO: 40);IDFILKISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIK CD8α hinge;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG CD8α TM;LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY 4-1BB signalingIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR domain;SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM CD3zeta signalingGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK domainGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 57) 32G8MALPVTALLLPLALLLHAARPEVNLVESGGGLVKPGGSL CD8α signal peptide;RLSCEASGFTFSYAWMSWVRQAPGKGLEWVGRIKSITDG 32G8 VH (Table 1, SEQGVIDYAAPVRNRCTISRDDSRNTLYLEMHSLKTEDTAVY ID NO: 43);YCTTIPGNDDFDMWGQGRMVTVSSGGGGSGGGGSGGGGS GS linker;GGGGSDIVLTQSPLSLSVTPGEPASISCRSSQSLLYSNG 32G8 VL (Table 1, SEQKNYLDWFLHKPGQSPQLLIYLGSNRASGVPDRFSGSGSG ID NO: 40);IDFILKISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIK CD8α hinge;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG CD8α TM;LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY 4-1BB signalingIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR domain;SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM CD3zeta signalingGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK domainGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 58) 26B9MALPVTALLLPLALLLHAARPEVQLVESWGVLVKPGGSL CD8α signal peptide;RLSCAASGFIFNNAWMSWVRQAPGKGLEWIGRIKSKSDG 26B9 VH (Table 1, SEQGTTDYAAPVKDRFTISRDDSKDTLYLQMNGLKTEDTAVY ID NO: 41);FCTTAPGGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GS linker;GGGSDIVLTQSPLSLPVTPGEPASISCRSSQSLLHRDGF 26B9 VL (Table 1, SEQNYLDWFLQKPGQSPQLLIYLASSRASGVPDRFSGSDSGT ID NO: 42);DFTLKISRVEAEDVGVYYCMQALQTPITFGQGTRLEIK CD8α hinge;TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG CD8α TM;LDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLY 4-1BB signalingIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSR domain;SADAPAYQQGQNQLYNELKLGRREEYDVLDKRRGRDPEM CD3zeta signalingGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK domainGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 59) 30D8MALPVTALLLPIALLLHAARPEVQLVESGGGLVKPGGSL CD8α signal peptide;RLSCEASGFTFSDAWMSWVRQAPGKGLEWVGRIKSKTDG 30D8 VH (Table 1, SEQGTTDYVVPLNGRFIISRDDSRNTLYLQLNNLKTEDTAVY ID NO: 37);YCTTVPGSYGYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GS linker;GGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHNKRMN 30D8 VL (Table 1, SEQYLDWFLQKPGQSPQLLIYLASNRASGVPDRFSGGGSGTD ID NO: 38);FTLKISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIKTT CD8α hinge;TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD CD8α TM;FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF 4-1BB signalingKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA domain;DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG CD3zeta signalingKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH domainDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 60) C6MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGSSV CD8α signal peptide;KVSCKASGDTFSSNAISWVRQAPGQGLEWMGVIIPIFGT C6 VH (Table 1, SEQ IDADYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC NO; 48);ARHTYHEYAGGYYGGAMDPWGQGTLVTVSSGGGGSGGGG GS linker;SGGGGSGGGGSELQSVLTQPPSASGTPGQRVTISCSGSS C6 VL (Table 1, SEQ IDSNIGSNYVYWYQQLPGTAPKILIYRNNQRPSGVPDRFSG NO: 49);SKSGTSASLAISGLRSEDEADYYCAAWDDNLSGWVFGTG CD8α hinge;TKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG CD8α TM;AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRG 4-1BB signalingRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL domain;RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR CD3zeta signalingGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG domainERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 61)

Table 5B provides nucleic acid sequences of exemplary scFvs of EGFRvIIIspecific CARs of the present invention. In Table 5B, the sequenceencoding the CD8α signal/leader peptide is underlined.

TABLE 53  Nucleic add sequences of exemplary EGFRvTTT specific scFvs CARscFv Nucleic Add Sequence h62G7-ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCCCTGGCTCTGCTGCTGCACG L6/EQCTGCTCGCCCTGATGTGGTCATGACTCAGTCTCCCCTGTCTCTGCCCGTCAC scFvCCTGGGACAGCCCGCCAGCATCTCCTGCAAGAGCTCCCAGAGCCTGCTGTACTCCAACGGCAAGACCTATCTGAATTGGTTCCAGCAGAGACCCGGCCAGAGCCCTCGGAGACTGATCTACCAGGTGTCTAAGCTGGACAGCGGCGTGCCTGATCGCTTCTCTGGAAGCGGATCCGGAACCGACTTTACACTGAAGATCAGCCGGGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCGGCCAGGATACCCACTTCCCACTGACATTTGGCGGCGGCACCAAGGTGGAGATCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCCAGGTGCAGCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCTGGCGCCTCCGTGAAGGTGTCTTGTAAGGCCAGCGGCTACACATTCACCGATTATACACTGCACTGGGTGCGGCAGGCCCCTGGCCAGGGACTGGAGTGGATGGGAGGAATCTGGCCTATCACCGGAGGAACCACATACAACCAGAAGTTTAAGGGCAGAGTGACAATGACCAGGGACACATCTACCAGCACAGTGTATATGGAGCTGTCTAGCCTGCGCTCCGAGGATACAGCCGTGTACTATTGCGCCAGAGGCGAGGCACAGGGATCTTGGGGACAGGGCACCCTGGTGACAGTGTCCTCT (SEQ ID NO: 228) 42G9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGGCCTCAGGTGACCCTGAAGGAGAGCGGCCCTGTGCTGCTGAAGCCAACAGAGACCCTGACACTGACCTGCACAGTGTCTGGCTTCAGCCTGTCCAACCCCCGGATGGGCGTGAGCTGGATCAGACAGCCCCCTGGCAAGGCCCTGGAGTGGTTCGCCCACATCTTTTCTACCGATGAGAAGAGCCTGAAGCTGTCCCTGAGATCTAGGCTGACCCTGAGCAAGGACACATCTAAGAGCCAGGTGGTGCTGACCATGACAAACATGGCCCCTGTGGACTCCGCCACATACTATTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGACTTTTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGCGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGCTCCGGCGGCGGCGGCTCCGAGGTGGTGCTGACCCAGAGCCCTGCCACACTGTCCGTGTCTCCAGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGCGCAGCAATCTGGCCTGGTACCAGCAGAAGTCCGGCCAGGCCCCAAGACTGCTGATCTATGGCTCCACCATCAGGGCCACAGGAGTGCCAGCACGCTTCTCTGGAAGCGGATCCGGCACAGAGTTTACCCTGACAATCTCCTCTCTGCAGTCCGAGGATTTCGCCGTGTACTATTGCCAGCAGTACTCTGACTGGCCCTTCACCTTTGGCCCTGGCACAAAGGTGGATATCAAG (SEQ ID NO: 229) 32A10ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGGCCACAGGTGACCCTGAAGGAGTCCGGCCCCGTGCTGGTGAAGCCTACAGAGACCCTGACACTGACCTGCACAGTGTCCGGCTTCTCTCTGAGCAACGCCCGCATGGGCGTGTCTTGGATCAGGCAGCCCCCTGGCAAGGCCCTGGAGTGGCTGGCCCACATCTTTTCCACCGACGAGAAGTCTATCCGGAGAAGCCTGCGCTCCAGGCTGACCCTGAGCAAGGATACATCCAAGTCTCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGATACCGCCACATACTTCTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTTGATTACTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGAGGTGGTCATGACCCAGAGCCCAGCCACACTGAGCGTGTCCCCTGGCGAGAGGGTGACCCTGTCCTGTAGGGCATCTCAGAGCGTGTCCTCTAACTTCGCCTGGTATCAGCAGAGACCAGGCCAGGCACCAAGGCTGCTGCTGTACGGAGCAACCACAAGAGCCACAGGACTGCCCGGCAGGTTTTCCGGATCTGGAAGCGGCACCGAGAATATCCTGACAATCAGCTCCCTGCAGTCTGAGGACTTCGCCATCTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCAGCAAGGTGGACATCAAG (SEQ ID NO: 230) 20B9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGACCTCAGGTGACCCTGAAGGAGTCCGGCCCTGTGCTGGTGAAGCCAACAGAGACCCTGACACTGACCTGCACAGTGTCTGGCTTCAGCCTGTCCAACGCAAGGATGGGCGTGAGCTGGATCAGGCAGCCCCCTGGCAAGGCCCTGGAGTGGCTGGGCCACATCTTTAGCACCGACGAGAAGTCTTACAGCACATCCCTGAGAGGCAGGATCACCATCTCTAAGGATACAAGCAGAGGCCTGGTGGTGCTGACCCTGACAAACATGGACCCCGTGGATACCGCCACATACTATTGCGCCAGGGACAGCTCCAATTACGAGGGCTATTTCGATTTTTGGGGCCCTGGCTTCCTGGTGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCCCCTGCCACACTGTCTGTGAGCCCAGGCGAGAGAGCCACCCTGTCTTGTAGGGTGTCCCAGTCTATCGGCGCCAATCTGGCCTGGTACCAGCAGAAGTTCGGCCAGGCCCCAAGGCTGCTGATCTATGGAGCATCCACCAGAGCCACAGGAATCCCCGTGAGGTTCTCCGGAGGAGGATCTGGAACCGAGTTTACCCTGACAATCTCCTCTCTGCAGAGCGAGGACTTTGCCATCTACTCCTGCCAGCAGTACATCTATTGGCCCTTCACATTTGGCCCTGGCACCACAGTGGATATCAAG (SEQ ID NO: 231) 14C11ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGACCACAGGTGACCCTGAAGGAGAGCGGACCCGTGCTGGTGAAGCCTACAGAGACCCTGACACTGACCTGCACAGTGAGCGGCTTCTCCCTGAACAATGCAAGGATGGGCGTGTCCTGGATCAGGCAGCCCCCTGGCAAGGCCCTGGAGTGGTTCGCCCACATCTTTAGCACCGACGAGAAGTCCTTTCGCACATCTCTGAGAAGCAGGCTGACCCTGAGCAAGGATACAAGCAAGTCCCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGGATACCGCCACATACTATTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGATTACTGGGGCCAGGGCATCCTGGTGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCTCCCGCCACACTGTCTGTGAGCCCTGGCGAGAGAGCCACACTGAGCTGTAGGGCCTCCCAGTCTGTGAGCAACAATCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCACCAAGGCTGCTGATCTACGGAGCATCCACCAGAGCCACAGGAGTGCCAGCAAGGTTCTCCGGATCTGACAGCGGCACCGAGTTTAGCCTGACAATCTCCTCTCTGCAGTCCGAGGACTTCGCCGTGTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCACAAAGGTGGAGATCAAG (SEQ ID NO: 232) 20E12ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scfvCAGCAACGCCAGAGGTGAACCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAGTGGGTGGGACGGATCAAGTCCATCGCAGACGGAGGAGCAACCGATTACGCAGCCCCTGTGAGAAACAGGTTCACAATCTCCAGAGACGATTCTAGGAATACCCTGTATCTGGAGATGCACTCTCTGAAGACAGAGGACACCGCCGTGTACTATTGCACCACAATCCCTGGCAACGACGCCTTTGATATGTGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCCGGCGAGCCTGCAAGCATCTCCTGTAGATCTAGCCAGAGCCTGCTGTACTCCAACGGCAAGAATTATCTGGATTGGTTCCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTGCCAGACCGGTTCTCTGGAAGCGGATCCGGCATCGACTTCATCCTGAAGATCAGCAGGGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAG (SEQ. ID NO: 233) 32G8ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGGCCAGAGGTGAACCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTCAGCTACGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAGTGGGTGGGCCGGATCAAGTCCATCACCGACGGAGGCGTGATCGATTACGCAGCACCTGTGAGAAAGAGGTGCACAATCTCCAGAGACGATTCTAGGAATACCCTGTATCTGGAGATGCACTCTCTGAAGACAGAGGACACCGCCGTGTACTATTGTACCACAATCCCTGGCAACGACGATTTCGATATGTGGGGCCAGGGCAGAATGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCCGGCGAGCCTGCAAGCATCTCCTGTAGGTCTAGCCAGAGCCTGCTGTACTCCAACGGCAAGAATTATCTGGATTGGTTTCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTGCCAGACCGGTTCTCTGGAAGCGGATCCGGCATCGACTTCATCCTGAAGATCAGCCGCGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAG (SEQ ID NO: 234) 26B9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGGCCAGAGGTGCAGCTGGTGGAGTCTTGGGGCGTGCTGGTGAAGCCTGGCGGATCTCTGAGGCTGAGCTGCGCAGCATCCGGCTTCATCTTTAACAATGCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGACTGGAGTGGATCGGCCGGATCAAGAGCAAGTCCGACGGAGGAACCACAGATTACGCAGCACCTGTGAAGGACCGCTTCACAATCTCTCGGGACGATAGCAAGGATACCCTGTATCTGCAGATGAACGGCCTGAAGACAGAGGACACCGCCGTGTACTTCTGCACCACAGCCCCTGGCGGCCCTTTTGATTATTGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGAGGAGGAGGAAGCGGCGGAGGAGGCAGCGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGACATCGTGCTGACACAGAGCCCTCTGTCCCTGCCAGTGACCCCCGGCGAGCCTGCCTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGGGACGGCTTCAATTACCTGGATTGGTTTCTGCAGAAGCCAGGCCAGTCCCCCCAGCTGCTGATCTATCTGGCCTCCTCTAGAGCCTCTGGCGTGCCAGACAGGTTCTCCGGCTCTGACAGCGGCACAGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAGGCCCTGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAG (SEQ ID NO: 235) 30D8ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCCCTGCTGCTGCACG scFvCAGCAAGGCCTGAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTGAGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTTAGCGACGCATGGATGTCCTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAGTGGGTGGGACGGATCAAGAGCAAGACAGACGGCGGCACCACAGATTACGTGGTGCCACTGAACGGCCGCTTCATCATCTCCCGCGACGATTCTCGGAATACCCTGTATCTGCAGCTGAACAATCTGAAGACAGAGGATACCGCCGTGTACTATTGCACCACAGTGCCAGGCTCCTACGGCTATTGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGCGGCGGCTCTGACATCGTGATGACACAGTCTCCACTGAGCCTGCCAGTGACCCCTGGCGAGCCAGCCTCCATCTCTTGTCGCTCTAGCCAGAGCCTGCTGCACAACAAGCGGAACAATTACCTGGATTGGTTTCTGCAGAAGCCTGGCCAGTCCCCTCAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAGACAGGTTCTCTGGAGGAGGAAGCGGAACAGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCTATCACCTTCGGCCAGGGCAAGACTGGAGATCAAG (SEQ ID NO: 236) C6 scFvATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCCCTGCTGCTGCACGCAGCAAGGCCACAGGTGCAGCTGGTGCAGTCCGGAGCAGAGGTGAAGAAGCCTGGCAGCTCCGTGAAGGTGAGCTGCAAGGCCTCCGGCGACACATTCTCTAGCAACGCAATCAGCTGGGTGCGCCAGGCCCCTGGCCAGGGACTGGAGTGGATGGGCGTGATCATCCCTATCTTCGGCACCGCCGACTATGCCCAGAAGTTTCAGGGCCGGGTGACAATCACCGCCGATGAGTCTACAAGCACCGCCTACATGGAGCTGTCCTCTCTGAGATCCGAGGACACAGCCGTGTACTATTGTGCCAGGCACACCTATCACGAGTACGCAGGAGGATACTATGGAGGAGCAATGGATCCTTGGGGACAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGCGGCGGCTCTGAGCTGCAGAGCGTGCTGACCCAGCCACCTTCCGCCTCTGGAACACCAGGCCAGAGGGTGACCATCAGCTGCTCCGGATCTAGCTCCAACATCGGCTCCAATTACGTGTATTGGTACCAGCAGCTGCCAGGCACAGCCCCCAAGATCCTGATCTACCGCAACAATCAGCGGCCTTCTGGCGTGCCAGATAGATTCTCTGGCAGCAAGTCCGGCACCTCTGCCAGCCTGGCAATCTCCGGCCTGAGGTCTGAGGACGAGGCCGATTACTATTGCGCCGCCTGGGACGATAACCTGAGCGGCTGGGTGTTTGGCACAGGCACCAAGCTGACAGTGCTG (SEQ ID NO: 237)

Table 5C provides exemplary nucleic acid sequences of exemplary EGFRvIIIspecific CARs of the present invention.

TABLE 5C  Nucleic acid sequences of exemplary EGFRvIII specific CARs CARCAR Nucleic Add Sequence Components h62G7-ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCCCTGGCT CD8α signal peptide; L6/EQCTGCTGCTGCACGCTGCTCGCCCTGATGTGGTCATGACT h62G7-L6/EQ VL (TableCAGTCTCCCCTGTCTCTGCCCGTCACCCTGGGACAGCCC 1 SEQ ID NO: 6);GCCAGCATCTCCTGCAAGAGCTCCCAGAGCCTGCTGTAC GS linker [(GGGGS)₄TCCAACGGCAAGACCTATCTGAATTGGTTCCAGCAGAGA (SEQ ID NO: 202)];CCCGGCCAGAGCCCTCGGAGACTGATCTACCAGGTGTCT h62G7-L6/EQ VHAAGCTGGACAGCGGCGTGCCTGATCGCTTCTCTGGAAGC (Table 1 SEQ ID NO: 5);GGATCCGGAACCGACTTTACACTGAAGATCAGCCGGGTG CD8α hinge;GAGGCAGAGGACGTGGGCGTGTACTATTGCGGCCAGGAT CD8α TM;ACCCACTTCCCACTGACATTTGGCGGCGGCACCAAGGTC 4-1BB signalingGAGATCAAGGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC domain;GGCGGCGGCGGCTCTGGCGGCGGCGGCAGCCAGGTGCAG CD3zeta signalingCTGGTGCAGAGCGGAGCAGAGGTGAAGAAGCCTGGCGCC domainTCCGTGAAGGTGTCTTGTAAGGCCAGCGGCTACACATTCACCGATTATACACTGCACTGGGTGCGGCAGGCCCCTGGCCAGGGACTGGAGTGGATGGGAGGAATCTGGCCTATCACCGGAGGAACCACATACAACCAGAAGTTTAAGGGCAGAGTGACAATGACCAGGGACACATCTACCAGCACAGTGTATATGGAGCTGTCTAGCCTGCGCTCCGAGGATACAGCCGTGTACTATTGCGCCAGAGGCGAGGCACAGGGATCTTGGGGACAGGGCACCCTGGTGACAGTGTCCTCTACCACAACCCCAGCACCAAGACCACCTACCCCTGCACCAACAATCGCCTCCCAGCCTCTGTCTCTGCGCCCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCAGAAGAAGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCCAGGTCTGCCGATGCACCAGCATACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCAGGCGCGAGGAGTATGACGTGCTGGATAAGAGGAGAGGAAGGGACCCTGAGATGGGAGGCAAGCCTAGGCGCAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGATAAGATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGCGAGCGGAGAAGGGGCAAGGGCCACGACGGGCTGTACCAGGGACTGTCAACCGCTACCAAGGATACTTACGACGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 238) 42G9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCTCAGGTGACCCTGAAG 42G9 VH (Table 1 SEQGAGAGCGGCCCTGTGCTGCTGAAGCCAACAGAGACCCTG ID NO: 9);ACACTGACCTGCACAGTGTCTGGCTTCAGCCTGTCCAAC GS linker;CCCCGGATGGGCGTGAGCTGGATCAGACAGCCCCCTGGC 42G9 VL (Table 1 SEQAAGGCCCTGGAGTGGTTCGCCCACATCTTTTCTACCGAT ID NO: 10);GAGAAGAGCCTGAAGCTGTCCCTGAGATCTAGGCTGACC CD8α hinge;CTGAGCAAGGACACATCTAAGAGCCAGGTGGTGCTGACC CD8α TM;ATGACAAACATGGCCCCTGTGGACTCCGCCACATACTAT 4-1BB signalingTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGAC domain;TTTTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGC cD3zeta signalingGGAGGAGGATCCGGAGGAGGAGGATCTGGCGGCGGCGGC domainTCCGGCGGCGGCGGCTCCGAGGTGGTGCTGACCCAGAGCCCTGCCACACTGTCCGTGTCTCCAGGCGAGAGAGCCACCCTGTCTTGTAGGGCCAGCCAGTCCGTGCGCAGCAATCTGGCCTGGTACCAGCAGAAGTCCGGCCAGGCCCCAAGACTGCTGATCTATGGCTCCACCATCAGGGCCACAGGAGTGCCAGCACGCTTCTCTGGAAGCGGATCCGGCACAGAGTTTACCCTGACAATCTCCTCTCTGCAGTCCGAGGATTTCGCCGTGTACTATTGCCAGCAGTACTCTGACTGGCCCTTCACCTTTGGCCCTGGCACAAAGGTGGATATCAAGACCACAACCCCTGCACCAAGGCCACCAACCCCAGCACCTACAATCGCAAGCCAGCCACTGTCCCTGAGACCCGAGGCCTGTAGGCCTGCAGCAGGAGGAGCAGTGCACACCCGCGGCCTGGACTTTGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACAACCCAGGAGGAGGATGGCTGCTCCTGTAGATTCCCTGAGGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCTCGGAGCGCCGACGCACCAGCATACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCACGGAGAAAGAACCCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGATAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGACGGACTGTACCAGGGACTGTCCACCGCAACAAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 239) 32A10ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCACAGGTGACCCTGAAG 32A10 VH (Table 1 SEQGAGTCCGGCCCCGTGCTGGTGAAGCCTACAGAGACCCTG ID NO: 11);ACACTGACCTGCACAGTGTCCGGCTTCTCTCTGAGCAAC GS linker;GCCCGCATGGGCGTGTCTTGGATCAGGCAGCCCCCTGGC 32A10 VL (Table 1 SEQAAGGCCCTGGAGTGGCTGGCCCACATCTTTTCCACCGAC ID NO: 12);GAGAAGTCTATCCGGAGAAGCCTGCGCTCCAGGCTGACC CD8α hinge;CTGAGCAAGGATACATCCAAGTCTCAGGTGGTGCTGACC CD8α TM;ATGACAAACATGGACCCCGTGGATACCGCCACATACTTC 4-1BB signalingTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTTGAT domain;TACTGGGGCCAGGGCACCCTGGTGACAGTGTCTAGCGGA CD3zeta signalingGGAGGAGGAAGCGGAGGAGGAGGATCTGGCGGCGGCGGC domainTCTGGCGGCGGCGGCAGCGAGGTGGTCATGACCCAGAGCCCAGCCACACTGAGCGTGTCCCCTGGCGAGAGGGTGACCCTGTCCTGTAGGGCATCTCAGAGCGTGTCCTCTAACTTCGCCTGGTATCAGCAGAGACCAGGCCAGGCACCAAGGCTGCTGCTGTACGGAGCAACCACAAGAGCCACAGGACTGCCCGGCAGGTTTTCCGGATCTGGAAGCGGCACCGAGAATATCCTGACAATCAGCTCCCTGCAGTCTGAGGACTTCGCCATCTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCAGCAAGGTGGACATCAAGACCACAACCCCTGCACCAAGACCACCAACCCCAGCACCTACAATCGCCTCTCAGCCTCTGAGCCTGCGCCCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACAAGGGGCCTGGACTTCGCCTGCGATATCTATATCTGGGCACCTCTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCCAGGTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAGGAAGGGATCCAGAGATGGGAGGCAAGCCTAGGCGCAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCTACCGCCACAAAGGACACCTACGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 240) 20B9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGACCTCAGGTGACCCTGAAG 20B9 VH (Table 1, SEQGAGTCCGGCCCTGTGCTGGTGAAGCCAACAGAGACCCTG ID NO: 13);ACACTGACCTGCACAGTGTCTGGCTTCAGCCTGTCCAAC GS linker;GCAAGGATGGGCGTGAGCTGGATCAGGCAGCCCCCTGGC 20B9 VL (Table 1, SEQAAGGCCCTGGAGTGGCTGGGCCACATCTTTAGCACCGAC ID NO: 14);GAGAAGTCTTACAGCACATCCCTGAGAGGCAGGATCACC CD8α hinge;ATCTCTAAGGATACAAGCAGAGGCCTGGTGGTGCTGACC CD8α TM;CTGACAAACATGGACCCCGTGGATACCGCCACATACTAT 4-1BB signalingTGCGCCAGGGACAGCTCCAATTACGAGGGCTATTTCGAT domain;TTTTGGGGCCCTGGCTTCCTGGTGACCGTGTCTAGCGGC CD3zeta signalingGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGA domainTCCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCCCCTGCCACACTGTCTGTGAGCCCAGGCGAGAGAGCCACCCTGTCTTGTAGGGTGTCCCAGTCTATCGGCGCCAATCTGGCCTGGTACCAGCAGAAGTTCGGCCAGGCCCCAAGGCTGCTGATCTATGGAGCATCCACCAGAGCCACAGGAATCCCCGTGAGGTTCTCCGGAGGAGGATCTGGAACCGAGTTTACCCTGACAATCTCCTCTCTGCAGAGCGAGGACTTTGCCATCTACTCCTGCCAGCAGTACATCTATTGGCCCTTCACATTTGGCCCTGGCACCACAGTGGATATCAAGACCACAACCCCTGCACCAAGGCCACCAACCCCAGCACCTACAATCGCAAGCCAGCCACTGTCCCTGAGACCAGAGGCATGTAGGCCTGCAGCAGGAGGAGCCGTGCACACCAGAGGCCTGGACTTTGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTAGCCGGTCCGCCGATGCACCAGCATACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGACCCAGAGATGGGAGGCAAGCCACGGAGAAAGAACCCCCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACCGCCACAAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 241) 14C11ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGACCACAGGTGACCCTGAAG 14C11 VH (Table 1 SEQGAGAGCGGACCCGTGCTGGTGAAGCCTACAGAGACCCTG ID NO: 15);ACACTGACCTGCACAGTGAGCGGCTTCTCCCTGAACAAT GS linker;GCAAGGATGGGCGTGTCCTGGATCAGGCAGCCCCCTGGC 14C11 VL (Table 1, SEQAAGGCCCTGGAGTGGTTCGCCCACATCTTTAGCACCGAC ID NO: 16);GAGAAGTCCTTTCGCACATCTCTGAGAAGCAGGCTGACC CD8α hinge;CTGAGCAAGGATACAAGCAAGTCCCAGGTGGTGCTGACC CD8α TM;ATGACAAACATGGACCCCGTGGATACCGCCACATACTAT 4-1BB signalingTGCGCCAGAGACAGCTCCAATTACGAGGGCTATTTCGAT domain;TACTGGGGCCAGGGCATCCTGGTGACCGTGTCTAGCGGC CD3zeta signalingGGCGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGA domainTCCGGCGGCGGCGGCTCTGAGATCGTGATGACCCAGTCTCCCGCCACACTGTCTGTGAGCCCTGGCGAGAGAGCCACACTGAGCTGTAGGGCCTCCCAGTCTGTGAGCAACAATCTGGCCTGGTATCAGCAGAAGCCAGGCCAGGCACCAAGGCTGCTGATCTACGGAGCATCCACCAGAGCCACAGGAGTGCCAGCAAGGTTCTCCGGATCTGACAGCGGCACCGAGTTTAGCCTGACAATCTCCTCTCTGCAGTCCGAGGACTTCGCCGTGTATTTTTGCCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCACAAAGGTGGAGATCAAGACCACAACCCCTGCACCAAGACCACCAACCCCAGCACCTACAATCGCATCCCAGCCTCTGTCTCTGAGACCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTATATCTGGGCACCTCTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGCAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTCGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCCCGGTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGATCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCTACCGCCACAAAGGACACCTACGATGCCCTGCATATGCAGGCACTGCCTCCAAGGTGA (SEQ ID NO: 242) 20E12ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCAGAGGTGAACCTGGTG 20E12 VH (Table 1, SEQGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTG ID NO: 39);AGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTCAGCTAC GS linker;GCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGA 20E12 VL (Table 1, SEQCTGGAGTGGGTGGGACGGATCAAGTCCATCGCAGACGGA ID NO: 40);GGAGCAACCGATTACGCAGCCCCTGTGAGAAACAGGTTC CD8α hinge;ACAATCTCCAGAGACGATTCTAGGAATACCCTGTATCTG CD8α TM;GAGATGCACTCTCTGAAGACAGAGGACACCGCCGTGTAC 4-1BB signalingTATTGCACCACAATCCCTGGCAACGACGCCTTTGATATG domain;TGGGGCCAGGGCACAATGGTGACCGTGAGCTCCGGCGGC CD3zeta signalingGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC domainGGGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCCGGCGAGCCTGCAAGCATCTCCTGTAGATCTAGCCAGAGCCTGCTGTACTCCAACGGCAAGAATTATCTGGATTGGTTCCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTGCCAGACCGGTTCTCTGGAAGCGGATCCGGCATCGACTTCATCCTGAAGATCAGCAGGGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAGACCACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCATCCCAGCCACTGTCTCTGAGGCCTGAGGCATGTCGGCCAGCAGCAGGAGGAGCAGTGCACACCCGCGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCTAGGAGCGCCGATGCACCAGCATACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTATGACGTGCTGGATAAGAGGAGGGGAAGGGACCCTGAGATGGGAGGCAAGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCT CCAAGGTGA (SEQ ID NO: 243) 32G8ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCAGAGGTGAACCTGGTG 32G8 VH (Table 1, SEQGAGTCCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTG ID NO: 43);AGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTCAGCTAC GS linker,GCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGA 32G8 VL (Table 1, SEQCTGGAGTGGGTGGGCCGGATCAAGTCCATCACCGACGGA ID NO: 40);GGCGTGATCGATTACGCAGCACCTGTGAGAAACAGGTGC CD8α hinge;ACAATCTCCAGAGACGATTCTAGGAATACCCTGTATCTG CD8α TM;GAGATGCACTCTCTGAAGACAGAGGACACCGCCGTGTAC 4-1BB signalingTATTGTACCACAATCCCTGGCAACGACGATTTCGATATG domain;TGGGGCCAGGGCAGAATGGTGACCGTGAGCTCCGGCGGC CD3zeta signalingGGCGGCTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGC domainGGGGGCGGCGGCTCTGACATCGTGCTGACACAGTCCCCACTGTCCCTGTCTGTGACCCCCGGCGAGCCTGCAAGCATCTCCTGTAGGTCTAGCCAGAGCCTGCTGTACTCCAACGGCAAGAATTATCTGGATTGGTTTCTGCACAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTACCTGGGATCTAATAGGGCAAGCGGAGTGCCAGACCGGTTCTCTGGAAGCGGATCCGGCATCGACTTCATCCTGAAGATCAGCCGCGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAGACCACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCATCCCAGCCACTGTCTCTGAGGCCTGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGAGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGCGCGGCCGGAAGAAGCTGCTGTATATCTTCAAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCTAGGAGCGCCGATGCACCAGCATACCAGCAGGGACAGAATCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTATGACGTGCTGGATAAGAGGAGGGGAAGGGATCCTGAGATGGGAGGCAAGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCT CCAAGGTGA (SEQ ID NO: 244) 26B9ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCAGAGGTGCAGCTGGTG 26B9 VH (Table 1, SEQGAGTCTTGGGGCGTGCTGGTGAAGCCTGGCGGATCTCTG ID NO: 41);AGGCTGAGCTGCGCAGCATCCGGCTTCATCTTTAACAAT GS linker;GCCTGGATGTCCTGGGTGCGCCAGGCCCCCGGCAAGGGA 26B9 VL (Table 1, SEQCTGGAGTGGATCGGCCGGATCAAGAGCAAGTCCGACGGA ID NO: 42);GGAACCACAGATTACGCAGCACCTGTGAAGGACCGCTTC CD8α hinge;ACAATCTCTCGGGACGATAGCAAGGATACCCTGTATCTG CD8α TM;CAGATGAACGGCCTGAAGACAGAGGACACCGCCGTGTAC 4-1BB signalingTTCTGCAGCACAGCCCCTGGCGGCCCTTTTGATTATTGG domain;GGCCAGGGCACACTGGTGACCGTGAGCTCCGGAGGAGGA CD3zeta signalingGGAAGCGGCGGAGGAGGCAGCGGCGGCGGCGGCTCTGGC domainGGCGGCGGCAGCGACATCGTGCTGACACAGAGCCCTCTGTCCCTGCCAGTGACCCCCGGCGAGCCTGCCTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGGGACGGCTTCAATTACCTGGATTGGTTTCTGCAGAAGCCAGGCCAGTCCCCCCAGCTGCTGATCTATCTGGCCTCCTCTAGAGCCTCTGGCGTGCCAGACAGGTTCTCCGGCTCTGACAGCGGCACAGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGATGTGGGCGTGTACTATTGCATGCAGGCCCTGCAGACACCCATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAGACCACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCATCCCAGCCACTGTCTCTGAGACCTGAGGCCTGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTCAAGCAGCCTTTTATGAGACCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCCCGGTCTGCCGATGCACCAGCATACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGCGCGGCAGAGATCCAGAGATGGGCGGCAAGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCA AGGTGA (SEQ ID NO: 245) 30D8ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCACTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCTGAGGTGCAGCTGGTG 30D8 VH (Table 1, SEQGAGAGCGGCGGCGGCCTGGTGAAGCCTGGCGGATCCCTG ID No: 37);AGGCTGTCTTGCGAGGCAAGCGGCTTCACCTTTAGCGAC GS linker;GCATGGATGTCCTGGGTGCGCCAGGCCCCTGGCAAGGGA 30D8 VL (Table 1, SEQCTGGAGTGGGTGGGACGGATCAAGAGCAAGACAGACGGC ID NO: 38);GGCACCACAGATTACGTGGTGCCACTGAACGGCCGCTTC CD8α hinge;ATCATCTCCCGCGACGATTCTCGGAATACCCTGTATCTG CD8α TM;CAGCTGAACAATCTGAAGACAGAGGATACCGCCGTGTAC 4-1BB signalingTATTGCACCACAGTGCCAGGCTCCTACGGCTATTGGGGC domain;CAGGGCACACTGGTGACCGTGAGCTCCGGCGGCGGCGGC CD3zeta signalingTCTGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGGGGC domainGGCGGCTCTGACATCGTGATGACACAGTCTCCACTGAGCCTGCCAGTGACCCCTGGCGAGCCAGCCTCCATCTCTTGTCGCTCTAGCCAGAGCCTGCTGCACAACAAGCGGAACAATTACCTGGATTGGTTTCTGCAGAAGCCTGGCCAGTCCCCTCAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAGACAGGTTCTCTGGAGGAGGAAGCGGAACAGACTTCACCCTGAAGATCAGCAGAGTGGAGGCCGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCTATCACCTTCGGCCAGGGCACAAGACTGGAGATCAAGACCACAACCCCAGCACCAAGGCCACCTACACCTGCACCAACCATCGCCTCCCAGCCTCTGTCTCTGAGACCAGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCTCTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGAGACCCGTGCAGACAACCCAGGAGGAGGACGGCTGCTCTTGTAGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTAGCCGGTCCGCCGATGCACCAGCATACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGATCCAGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGAGCACAGCCACCAAGGACACCTATGATGCCCTGCATATGCAGGCACTGCCTCCAAGG TGA (SEQ ID NO: 246) C6ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCC CD8α signal peptide;CTGCTGCTGCACGCAGCAAGGCCACAGGTGCAGCTGGTG C6 VH (Table 1, SEQ IDCAGTCCGGAGCAGAGGTGAAGAAGCCTGGCAGCTCCGTG NO: 48);AAGGTGAGCTGCAAGGCCTCCGGCGACACATTCTCTAGC GS linker;AACGCAATCAGCTGGGTGCGCCAGGCCCCTGGCCAGGGA 6C VL (Table 1, SEQ IDCTGGAGTGGATGGGCGTGATCATCCCTATCTTCGGCACC NO: 49);GCCGACTATGCCCAGAAGTTTCAGGGCCGGGTGACAATC CD8α hinge;ACCGCCGATGAGTCTACAAGCACCGCCTACATGGAGCTG CD8α TM;TCCTCTCTGAGATCCGAGGACACAGCCGTGTACTATTGT 4-1BB signalingGCCAGGCACACCTATCACGAGTACGCAGGAGGATACTAT domain;GGAGGAGCAATGGATCCTTGGGGACAGGGCACACTGGTG CD3zeta signalingACCGTGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGA domainAGCGGAGGAGGAGGAAGCGGGGGCGGCGGCTCTGAGCTGCAGAGCGTGCTGACCCAGCCACCTTCCGCCTCTGGAACACCAGGCCAGAGGGTGACCATCAGCTGCTCCGGATCTAGCTCCAACATCGGCTCCAATTACGTGTATTGGTACCAGCAGCTGCCAGGCACAGCCCCCAAGATCCTGATCTACCGCAACAATCAGCGGCCTTCTGGCGTGCCAGATAGATTCTCTGGCAGCAAGTCCGGCACCTCTGCCAGCCTGGCAATCTCCGGCCTGAGGTCTGAGGACGAGGCCGATTACTATTGCGCCGCCTGGGACGATAACCTGAGCGGCTGGGTGTTTGGCACAGGCACCAAGCTGACAGTGCTGACCAGAACCCCTGCACCAAGACCACCAACACCAGCACCTACCATCGCAAGCCAGCCACTGTCCCTGAGACCCGAGGCCTGTAGGCCTGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTTATGCGCCCTGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGAGGGTGAAGTTTTCCCGGTCTGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGGGACCCTGAGATGGGAGGCAAGCCACGGAGAAAAACCCCCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCCCTGC ATATGCAGGCACTGCCTCCAAGGTGA(SEQ. ID NO: 247)

Downregulation or mutation of target antigens is commonly observed incancer cells, creating antigen-loss escape variants. Thus, to offsettumor escape and render immune cell more specific to target, theEGFRvIII specific CAR can comprise one or more additional extracellularligand-binding domains, to simultaneously bind different elements intarget thereby augmenting immune cell activation and function. In oneembodiment, the extracellular ligand-binding domains can be placed intandem on the same transmembrane polypeptide, and optionally can beseparated by a linker. In some embodiments, said different extracellularligand-binding domains can be placed on different transmembranepolypeptides composing the CAR. In some embodiments, the inventionrelates to a population of CARs, each CAR comprising a differentextracellular ligand-binding domain. In a particular, the inventionrelates to a method of engineering immune cells comprising providing animmune cell and expressing at the surface of the cell a population ofCARs, each CAR comprising different extracellular ligand-bindingdomains. In another particular embodiment, the invention relates to amethod of engineering an immune cell comprising providing an immune celland introducing into the cell polynucleotides encoding polypeptidescomposing a population of CARs each one comprising differentextracellular ligand-binding domains. By population of CARs, it is meantat least two, three, four, five, six or more CARs each one comprisingdifferent extracellular ligand-binding domains. The differentextracellular ligand-binding domains according to the invention canpreferably simultaneously bind different elements in target therebyaugmenting immune cell activation and function. The invention alsorelates to an isolated immune cell which comprises a population of CARseach one comprising different extracellular ligand-binding domains.

In another aspect, the invention provides polynucleotides encoding anyof the CARs and polypeptides described herein. Polynucleotides can bemade and expressed by procedures known in the art.

In another aspect, the invention provides compositions (such as apharmaceutical compositions) comprising any of the cells of theinvention. In some embodiments, the composition comprises a cellcomprising a polynucleotide encoding any of the CARs described herein.

Expression vectors, and administration of polynucleotide compositionsare further described herein.

In another aspect, the invention provides a method of making any of thepolynucleotides described herein.

Polynucleotides complementary to any such sequences are also encompassedby the invention. Polynucleotides may be single-stranded (coding orantisense) or double-stranded, and may be DNA (genomic, cDNA orsynthetic) or RNA molecules. RNA molecules include HnRNA molecules,which contain introns and correspond to a DNA molecule in a one-to-onemanner, and mRNA molecules, which do not contain introns. Additionalcoding or non-coding sequences may, but need not, be present within apolynucleotide of the invention, and a polynucleotide may, but need not,be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes an antibody or a portion thereof) or may comprisea variant of such a sequence. Polynucleotide variants contain one ormore substitutions, additions, deletions and/or insertions such that theimmunoreactivity of the encoded polypeptide is not diminished, relativeto a native immunoreactive molecule. The effect on the immunoreactivityof the encoded polypeptide may generally be assessed as describedherein. Variants preferably exhibit at least about 70% identity, morepreferably, at least about 80% identity, yet more preferably, at leastabout 90% identity, and most preferably, at least about 95% identity toa polynucleotide sequence that encodes a native antibody or a portionthereof.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, or 40 to about 50, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e. the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a nativeantibody (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ duringhybridization a denaturing agent, such as formamide, for example, 50%(v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the invention. Further,alleles of the genes comprising the polynucleotide sequences providedherein are within the scope of the invention. Alleles are endogenousgenes that are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

The polynucleotides of this invention can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, supra, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructsthat contain a polynucleotide according to the invention. It is impliedthat an expression vector must be replicable in the host cells either asepisomes or as an integral part of the chromosomal DNA. Suitableexpression vectors include but are not limited to plasmids, viralvectors, including adenoviruses, adeno-associated viruses, retroviruses,cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (i.e., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introducedinto the host cell by any of a number of appropriate means, includingelectroporation, transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (e.g., where thevector is an infectious agent such as vaccinia virus). The choice ofintroducing vectors or polynucleotides will often depend on features ofthe host cell.

A polynucleotide encoding an EGFRvIII specific CAR disclosed herein mayexist in an expression cassette or expression vector (e.g., a plasmidfor introduction into a bacterial host cell, or a viral vector such as abaculovirus vector for transfection of an insect host cell, or a plasmidor viral vector such as a lentivirus for transfection of a mammalianhost cell). In some embodiments, a polynucleotide or vector can includea nucleic acid sequence encoding ribosomal skip sequences such as, forexample without limitation, a sequence encoding a 2A peptide. 2Apeptides, which were identified in the Aphthovirus subgroup ofpicomaviruses, causes a ribosomal “skip” from one codon to the nextwithout the formation of a peptide bond between the two amino acidsencoded by the codons (see (Donnelly and Elliott 2001; Atkins, Wills etal. 2007; Doronina, Wu et al. 2008)). By “codon” is meant threenucleotides on an mRNA (or on the sense strand of a DNA molecule) thatare translated by a ribosome into one amino acid residue. Thus, twopolypeptides can be synthesized from a single, contiguous open readingframe within an imRNA when the polypeptides are separated by a 2Aoligopeptide sequence that is in frame. Such ribosomal skip mechanismsare well known in the art and are known to be used by several vectorsfor the expression of several proteins encoded by a single messengerRNA.

To direct transmembrane polypeptides into the secretory pathway of ahost cell, in some embodiments, a secretory signal sequence (also knownas a signal peptide, leader sequence, prepro sequence or pre sequence)is provided in a polynucleotide sequence or vector sequence. Thesecretory signal sequence is operably linked to the transmembranenucleic acid sequence, i.e., the two sequences are joined in the correctreading frame and positioned to direct the newly synthesized polypeptideinto the secretory pathway of the host cell. Secretory signal sequencesare commonly positioned 5′ to the nucleic acid sequence encoding thepolypeptide of interest, although certain secretory signal sequences maybe positioned elsewhere in the nucleic acid sequence of interest (see,e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat.No. 5,143,830). In some embodiments the signal peptide comprises theamino acid sequence shown in SEQ ID NO: 206 or 214. Those skilled in theart will recognize that, in view of the degeneracy of the genetic code,considerable sequence variation is possible among these polynucleotidemolecules. In some embodiments, nucleic acid sequences of the inventionare codon-optimized for expression in mammalian cells, preferably forexpression in human cells. Codon-optimization refers to the exchange ina sequence of interest of codons that are generally rare in highlyexpressed genes of a given species by codons that are generally frequentin highly expressed genes of such species, such codons encodingidentical amino acids as the codons that are being exchanged.

Methods of Engineering an Immune Cell

Methods of preparing immune cells for use in immunotherapy are providedherein. In some embodiments, the methods comprise introducing a CARaccording to the invention into immune cells, and expanding the cells.In some embodiments, the invention relates to a method of engineering animmune cell comprising: providing a cell and expressing at the surfaceof the cell at least one CAR as described above. Methods for engineeringimmune cells are described in, for example, PCT Patent ApplicationPublication Nos. WO/2014/039523, WO/2014/184741, WO/2014/191128,WO/2014/184744, and WO/2014/184143, each of which is incorporated hereinby reference in its entirety. In some embodiments, the method comprises:transforming the cell with at least one polynucleotide encoding CAR asdescribed above, and expressing the polynucleotides in the cell.

In some embodiments, the polynucleotides are present in lentiviralvectors for stable expression in the cells.

In some embodiments, the method can further comprise a step ofgenetically modifying a cell by inactivating at least one geneexpressing, for example without limitation, a component of the TCR, atarget for an immunosuppressive agent, an HLA gene, and/or an immunecheckpoint protein such as, for example, PDCD1 or CTLA-4. Byinactivating a gene it is intended that the gene of interest is notexpressed in a functional protein form. In some embodiments, the gene tobe inactivated is selected from the group consisting of, for examplewithout limitation, TCRα, TCRβ, dCK, CD52, GR, PD-1, and CTLA-4. In someembodiments the method comprises inactivating one or more genes byintroducing into the cells a rare-cutting endonuclease able toselectively inactivate a gene by selective DNA cleavage. In someembodiments the rare-cutting endonuclease can be, for example, atranscription activator-like effector nuclease (TALE-nuclease) or Cas9endonuclease.

In some embodiments, an additional catalytic domain is used with arare-cutting endonuclease to enhance its capacity to inactivate targetedgenes. For example, an additional catalytic domain can be a DNAend-processing enzyme. Non-limiting examples of DNA end-processingenzymes include 5-3′ exonucleases, 3-5′ exonucleases, 5-3′ alkalineexonucleases, 5′ flap endonucleases, helicases, hosphatase, hydrolasesand template-independent DNA polymerases. Non-limiting examples of suchcatalytic domain comprise of a protein domain or catalytically activederivate of the protein domain selected from the group consisting ofhExoI (EXO1_HUMAN), Yeast ExoI (EXO1_YEAST), E. coli ExoI, Human TREX2,Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminaldeoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST). Insome embodiments, an additional catalytic domain can have a3′-5′-exonuclease activity, and in some embodiments, said additionalcatalytic domain is TREX, more preferably TREX2 catalytic domain(WO2012/058458). In some embodiments, said catalytic domain is encodedby a single chain TREX polypeptide. The additional catalytic domain maybe fused to a nuclease fusion protein or chimeric protein. In someembodiments, the additional catalytic domain is fused using, forexample, a peptide linker.

In some embodiments, the method further comprises a step of introducinginto cells an exogeneous nucleic acid comprising at least a sequencehomologous to a portion of the target nucleic acid sequence, such thathomologous recombination occurs between the target nucleic acid sequenceand the exogeneous nucleic acid. In some embodiments, said exogenousnucleic acid comprises first and second portions which are homologous toregion 5′ and 3′ of the target nucleic acid sequence, respectively. Theexogenous nucleic acid may also comprise a third portion positionedbetween the first and the second portion which comprises no homologywith the regions 5′ and 3′ of the target nucleic acid sequence.Following cleavage of the target nucleic acid sequence, a homologousrecombination event is stimulated between the target nucleic acidsequence and the exogenous nucleic acid. In some embodiments, homologoussequences of at least about 50 bp, greater than about 100 bp, or greaterthan about 200 bp can be used within the donor matrix. The exogenousnucleic acid can be, for example without limitation, from about 200 bpto about 6000 bp, more preferably from about 1000 bp to about 2000 bp.Shared nucleic acid homologies are located in regions flanking upstreamand downstream the site of the break, and the nucleic acid sequence tobe introduced is located between the two arms.

In some embodiments, a nucleic acid successively comprises a firstregion of homology to sequences upstream of said cleavage; a sequence toinactivate a targeted gene selected from the group consisting of TCRα,TCRβ, CD52, glucocorticoid receptor (GR), deoxycytidine kinase (dCK),and an immune checkpoint protein such as for example programmed death-1(PD-1); and a second region of homology to sequences downstream of thecleavage. The polynucleotide introduction step can be simultaneous,before or after the introduction or expression of the rare-cuttingendonuclease. Depending on the location of the target nucleic acidsequence wherein break event has occurred, such exogenous nucleic acidcan be used to knock-out a gene, e.g. when exogenous nucleic acid islocated within the open reading frame of the gene, or to introduce newsequences or genes of interest. Sequence insertions by using suchexogenous nucleic acid can be used to modify a targeted existing gene,by correction or replacement of the gene (allele swap as a non-limitingexample), or to up- or down-regulate the expression of the targeted gene(promoter swap as non-limiting example), the targeted gene correction orreplacement. In some embodiments, inactivation of a genes selected fromthe group consisting of TCRα, TCRβ, CD52, GR, dCK, and immune checkpointproteins, can be done at a precise genomic location targeted by aspecific TALE-nuclease, wherein said specific TALE-nuclease catalyzes acleavage and wherein the exogenous nucleic acid successively comprisingat least a region of homology and a sequence to inactivate one targetedgene selected from the group consisting of TCRα, TCRβ, CD52, GR, dCK,immune checkpoint proteins which is integrated by homologousrecombination. In some embodiments, several genes can be, successivelyor at the same time, inactivated by using several TALE-nucleasesrespectively and specifically targeting one defined gene and severalspecific polynucleotides for specific gene inactivation.

In some embodiments, the method comprises inactivation of one or moreadditional genes selected from the group consisting of TCRα, TCRβ, CD52,GR, dCK, and immune checkpoint proteins. In some embodiments,inactivation of a gene can be accomplished by introducing into the cellsat least one rare-cutting endonuclease such that the rare-cuttingendonuclease specifically catalyzes cleavage in a targeted sequence ofthe cell genome; and optionally, introducing into the cells an exogenousnucleic acid successively comprising a first region of homology tosequences upstream of the cleavage, a sequence to be inserted in thegenome of the cell, and a second region of homology to sequencesdownstream of the cleavage; wherein the introduced exogenous nucleicacid inactivates a gene and integrates at least one exogenouspolynucleotide sequence encoding at least one recombinant protein ofinterest. In some embodiments, the exogenous polynucleotide sequence isintegrated within a gene encoding a protein selected from the groupconsisting of TCRα, TCRβ, CD52, GR, dCK, and immune checkpoint protein.

In another aspect, a step of genetically modifying cells can comprise:modifying T cells by inactivating at least one gene expressing a targetfor an immunosuppressive agent, and; expanding the cells, optionally inpresence of the immunosuppressive agent. An immunosuppressive agent isan agent that suppresses immune function by one of several mechanisms ofaction. An immunosuppressive agent can diminish the extent and/orvoracity of an immune response. Non-limiting examples ofimmunosuppressive agents include calcineurin inhibitors, targets ofrapamycin, interleukin-2 α-chain blockers, inhibitors of inosinemonophosphate dehydrogenase, inhibitors of dihydrofolic acid reductase,corticosteroids, and immunosuppressive antimetabolites. Some cytotoxicimmunosuppressants act by inhibiting DNA synthesis. Others may actthrough activation of T cells or by inhibiting the activation of helpercells. The methods according to the invention allow conferringimmunosuppressive resistance to T cells for immunotherapy byinactivating the target of the immunosuppressive agent in T cells. Asnon-limiting examples, targets for immunosuppressive agent can be areceptor for an immunosuppressive agent such as for example withoutlimitation CD52, glucocorticoid receptor (GR), FKBP family gene members,and cyclophilin family gene members.

In some embodiments, the genetic modification of the method involvesexpression, in provided cells to engineer, of one rare-cuttingendonuclease such that the rare-cutting endonuclease specificallycatalyzes cleavage in one targeted gene thereby inactivating thetargeted gene. In some embodiments, a method of engineering cellscomprises at least one of the following steps: providing a T cell, suchas from a cell culture or from a blood sample; selecting a gene in the Tcell expressing a target for an immunosuppressive agent; introducinginto the T cell a rare-cutting endonuclease able to selectivelyinactivate by DNA cleavage, preferably by double-strand break the geneencoding a target for the immunosuppressive agent, and expanding thecells, optionally in presence of the immunosuppressive agent.

In some embodiments, the method comprises: providing a T cell, such asfrom a cell culture or from a blood sample; selecting a gene in the Tcell wherein the gene expresses a target for an immunosuppressive agent;transforming the T cell with nucleic acid encoding a rare-cuttingendonuclease able to selectively inactivate by DNA cleavage, preferablyby double-strand break the gene encoding a target for theimmunosuppressive agent, and expressing the rare-cutting endonucleasesinto the T cells; and expanding the cells, optionally in presence of theimmunosuppressive agent.

In some embodiments, the rare-cutting endonuclease specifically targetsCD52 or GR. In some embodiments, the gene selected for inactivationencodes CD52, and the immunosuppressive treatment comprises a humanizedantibody targeting CD52 antigen. In some embodiments, the gene selectedfor inactivation encodes GR, and the immunosuppressive treatmentcomprises a corticosteroid such as dexamethasone. In some embodiments,the gene selected for inactivation is a FKBP family gene member or avariant thereof and the immunosuppressive treatment comprises FK506,also known as Tacrolimus or fujimycin. In some embodiments, the FKBPfamily gene member is FKBP12 or a variant thereof. In some embodiments,gene selected for inactivation is a cyclophilin family gene member or avariant thereof and the immunosuppressive treatment comprisescyclosporine.

In some embodiments, the rare-cutting endonuclease can be, for example,a meganuclease, a zinc finger nuclease, or a TALE-nuclease. In someembodiments, the rare-cutting endonuclease is a TALE-nuclease.

Also provided herein are methods of engineering T cells, suitable forimmunotherapy, wherein the methods comprise: genetically modifying Tcells by inactivating at least one immune checkpoint protein. In someembodiments the immune checkpoint protein is, for example, PD-1 and/orCTLA-4. In some embodiments, methods of genetically modifying a cellcomprises: modifying T cells by inactivating at least one immunecheckpoint protein; and expanding the cells. Immune checkpoint proteinsinclude, but are not limited to Programmed Death 1 (PD-1, also known asPDCD1 or CD279, accession number: NM_-005018), Cytotoxic T-LymphocyteAntigen 4 (CTLA-4, also known as CD152, GenBank accession numberAF414120.1), LAG3 (also known as CD223, accession number NM_002286.5),Tim3 (also known as HAVCR2, GenBank accession number JX049979.1), BTLA(also known as CD272, accession number NM_181780.3), BY55 (also known asCD160, GenBank accession number: CR541888.1), TIGIT (also known asVSTM3, accession number: NM_173799), B7H5 (also known as C10orf54,homolog of mouse vista gene, accession number: NM_022153.1), LAIR1 (alsoknown as CD305, GenBank accession number: CR542051.1), SIGLEC10(GeneBank accession number AY358337.1), 2B4 (also known as CD244,accession number: NM_001166664.1), which directly inhibit immune cells.For example, CTLA-4 is a cell-surface protein expressed on certain CD4and CD8 T cells; when engaged by its ligands (87-1 and B7-2) on antigenpresenting cells, T cell activation and effector function are inhibited.

In some embodiments, said method to engineer cells comprises at leastone of the following steps: providing a T cell, such as from a cellculture or from a blood sample; introducing into the T cell arare-cutting endonuclease able to selectively inactivate by DNAcleavage, preferably by double-strand break one gene encoding an immunecheckpoint protein; and expanding the cells. In some embodiments, themethod comprises: providing a T cell, such as from a cell culture orfrom a blood sample; transfecting said T cell with nucleic acid encodinga rare-cutting endonuclease able to selectively inactivate by DNAcleavage, preferably by double-strand break a gene encoding an immunecheckpoint protein; expressing the rare-cutting endonucleases into the Tcells; expanding the cells. In some embodiments, the rare-cuttingendonuclease specifically targets a gene selected from the groupconsisting of: PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1,SIGLEC10, 2B4, TCRα, and TCRβ. In some embodiments, the rare-cuttingendonuclease can be a meganuclease, a zinc finger nuclease or aTALE-nuclease. In some embodiments, the rare-cutting endonuclease is aTALE-nuclease.

In some embodiments, the present invention can be particularly suitablefor allogeneic immunotherapy. In such embodiments, cells may be modifiedby a method comprising: inactivating at least one gene encoding acomponent of the T cell receptor (TCR) in T cells; and expanding the Tcells. In some embodiments, the genetic modification of the methodrelies on the expression, in provided cells to engineer, of onerare-cutting endonuclease such that the rare-cutting endonucleasespecifically catalyzes cleavage in one targeted gene therebyinactivating the targeted gene. In some embodiments, said method toengineer cells comprises at least one of the following steps: providinga T cell, such as from a cell culture or from a blood sample;introducing into the T cell a rare-cutting endonuclease able toselectively inactivate by DNA cleavage, preferably by double-strandbreak at least one gene encoding a component of the T cell receptor(TCR), and expanding the cells.

In some embodiments, the method comprises: providing a T cell, such asfrom a cell culture or from a blood sample; transfecting said T cellwith nucleic acid encoding a rare-cutting endonuclease able toselectively inactivate by DNA cleavage, preferably by double-strandbreak at least one gene encoding a component of the T cell receptor(TCR); expressing the rare-cutting endonucleases into the T cells;sorting the transfected T cells, which do not express TCR on their cellsurface; and expanding the cells.

In some embodiments, the rare-cutting endonuclease can be ameganuclease, a zinc finger nuclease or a TALE-nuclease. In someembodiments, the rare-cutting endonuclease is a TALE-nuclease. In someembodiments the TALE-nucleases recognize and cleave a sequence encodingTCRα or TCRβ. In some embodiments a TALE-nuclease comprises apolypeptide sequence selected from the amino acid sequence shown in SEQID NO: 218, 219, 220, 221, 222, 223, 224, or 225.

TALE-nuclease polypeptide sequences: Repeat TRAC T01-L (SEQ ID NO: 218)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRAC T01-R (SEQ ID NO: 219)LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC T01-L (SEQ ID NO: 220)LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC T01-R (SEQ ID NO: 221)NPQRSTVWYLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC T02-L(SEQ ID NO: 222) LTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat TRBC T02-R (SEQ ID NO: 223)LTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat CD52 T02-L (SEQ ID NO: 224)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPQQVVAIASNGGGRPALE Repeat CD52 T02-R (SEQ ID NO: 225)LTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPEQVVAIASNIGGKQALETVQALLPVLCQAHGLTPEQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPQQVVAIASNGGGRPALE

In another aspect, another step of genetically modifying a cell can be amethod of expanding TCRα deficient T cells comprising introducing intothe T cell pTα (also known as preTCRα) or a functional variant thereofand expanding the cells, optionally through stimulation of the CD3complex. In some embodiments, the method comprises: a) transforming thecells with a nucleic acid encoding at least a fragment of pTα to supportCD3 surface expression; b) expressing said pTα into the cells; and c)expanding the cells, optionally through stimulation of the CD3 complex.

Also provided are methods of preparing T cells for immunotherapycomprising steps of a method provided herein for expansion of T cells.In some embodiments, the pTα polynucleotide sequence can be introducedrandomly or by homologous recombination. In some embodiments, theinsertion can be associated with the inactivation of the TCRα gene.

Different functional variants of pTα can be used. A “functional variant”of the peptide refers to a molecule substantially similar to either theentire peptide or a fragment thereof. A “fragment” of the pTα orfunctional variant thereof refers to any subset of the molecule, thatis, a shorter peptide than the full-length pTα. In some embodiments, pTαor functional variants can be, for example, full-length pTα or aC-terminal truncated pTα version. C-terminal truncated pTα lacks inC-terminal end one or more residues. As non limiting examples,C-terminal truncated pTα version lacks 18, 48, 62, 78, 92, 110 or 114residues from the C-terminus of the protein. Amino acid sequencevariants of the peptide can be prepared by mutations in the DNA whichencodes the peptide. Such functional variants include, for example,deletions from, or insertions or substitutions of, residues within theamino acid sequence. Any combination of deletion, insertion, andsubstitution may also be made to arrive at the final construct, providedthat the final construct possesses the desired activity, in particularthe restoration of a functional CD3 complex. In a preferred embodiment,at least one mutation is introduced in the different pTα versions asdescribed above to affect dimerization. As a non limiting example, amutated residue can be at least W46R, D22A, K24A, R102A or R117A of thehuman pTα protein or aligned positions using CLUSTALW method on pTαfamily or homologue member. Preferably pTα or a variant thereof asdescribed above comprise the mutated residue W46R or the mutatedresidues D22A, K24A, R102A and R117A. In some embodiments, said pTα orvariants are also fused to a signal-transducing domain such as CD28,OX40, ICOS, CD27, CD137 (4-1BB) and CD8 as non limiting examples. Theextracellular domain of pTα or variants as described above can be fusedto a fragment of the TCRα protein, particularly the transmembrane andintracellular domain of TCRα. pTα variants can also be fused to theintracellular domain of TCRα.

In some embodiments, pTα versions can be fused to an extracellularligand-binding domain. In some embodiments, pTα or a functional variantthereof is fused to a single chain antibody fragment (scFv) comprisingthe light and the heavy variable fragment of a target antigen specificmonoclonal antibody joined by a flexible linker.

The term “TCRα deficient T cell” refers to an isolated T cell that lacksexpression of a functional TCRα chain. This may be accomplished bydifferent means, as non limiting examples, by engineering a T cell suchthat it does not express any functional TCRα on its cell surface or byengineering a T cell such that it produces very little functional TCRαchain on its surface or by engineering a T cell to express mutated ortruncated form of TCRα chain. TCRα deficient cells can no longer beexpanded through CD3 complex. Thus, to overcome this problem and toallow proliferation of TCRα deficient cells, pTα or functional variantthereof is introduced into the cells, thus restoring a functional CD3complex. In some embodiments, the method further comprises introducinginto said T cells rare-cutting endonucleases able to selectivelyinactivate by DNA cleavage one gene encoding one component of the T cellreceptor (TCR). In some embodiments, the rare-cutting endonuclease is aTALE-nuclease.

In another aspect, engineered T cells obtained by the methods describedherein can be contacted with bispecific antibodies. For example, the Tcells can be contacted with bispecific antibodies ex vivo prior toadministration to a patient, or in vivo following administration to apatient. Bispecific antibodies comprise two variable regions withdistinct antigen properties that facilitate bringing the engineeredcells into proximity to a target antigen. As a non-limiting example, abispecific antibody can be directed against a tumor marker andlymphocyte antigen, such as for example without limitation CD3, and hasthe potential to redirect and activate any circulating T cells againsttumors.

In some embodiments, polynucleotides encoding polypeptides according tothe present invention can be mRNA which is introduced directly into thecells, for example by electroporation. In some embodiments, cytoPulsetechnology can be used to transiently permeabilize living cells fordelivery of material into the cells. Parameters can be modified in orderto determine conditions for high transfection efficiency with minimalmortality.

Also provided herein are methods of transforming T cells. In someembodiments, the method comprises: contacting a T cell with RNA andapplying to the T cell an agile pulse sequence consisting of: (a) anelectrical pulse with a voltage range from about 2250 to 3000 V percentimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about0.2 to 10 ms between the electrical pulses of step (a) and (b); (d) anelectrical pulse with a voltage range from about 2250 to 3000 V with apulse width of about 100 ms and a pulse interval of about 100 ms betweenthe electrical pulse of step (b) and the first electrical pulse of step(c); and (e) four electrical pulses with a voltage of about 325 V with apulse width of about 0.2 ms and a pulse interval of 2 ms between each of4 electrical pulses. In some embodiments, a method of transforming Tcell comprising contacting said T cell with RNA and applying to T cellan agile pulse sequence comprising: (a) an electrical pulse with avoltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450,2500, 2600, 2700, 2800, 2900 or 3000V per centimeter; (b) a pulse widthof 0.1 ms; (c) and a pulse interval of about 0.2, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b); (d)one electrical pulse with a voltage range from about 2250, of 2250,2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800,2900 or 3000V with a pulse width of 100 ms and a pulse interval of 100ms between the electrical pulse of step (b) and the first electricalpulse of step (c); and (e) 4 electrical pulses with a voltage of about325 V with a pulse width of about 0.2 ms and a pulse interval of about 2ms between each of 4 electrical pulses. Any values included in the valuerange described above are disclosed in the present application.Electroporation medium can be any suitable medium known in the art. Insome embodiments, the electroporation medium has conductivity in a rangespanning about 0.01 to about 1.0 milliSiemens.

In some embodiments, as non limiting examples, an RNA encodes arare-cutting endonuclease, one monomer of a rare-cutting endonucleasesuch as half-TALE-nuclease, a CAR, at least one component of amulti-chain chimeric antigen receptor, a pTα or functional variantthereof, an exogenous nucleic acid, and/or one additional catalyticdomain.

Engineered Immune Cells

The invention also provides engineered immune cells comprising any ofthe CAR polynucleotides described herein. In some embodiments, a CAR canbe introduced into an immune cell as a transgene via a plasmid vector.In some embodiments, the plasmid vector can also contain, for example, aselection marker which provides for identification and/or selection ofcells which received the vector.

CAR polypeptides may be synthesized in situ in the cell afterintroduction of polynucleotides encoding the CAR polypeptides into thecell. Alternatively, CAR polypeptides may be produced outside of cells,and then introduced into cells. Methods for introducing a polynucleotideconstruct into cells are known in the art. In some embodiments, stabletransfection methods can be used to integrate the polynucleotideconstruct into the genome of the cell. In other embodiments, transienttransfection methods can be used to transiently express thepolynucleotide construct, and the polynucleotide construct is notintegrated into the genome of the cell. In other embodiments,virus-mediated methods can be used. The polynucleotides may beintroduced into a cell by any suitable means such as for example,recombinant viral vectors (e.g. retroviruses, adenoviruses), liposomes,and the like. Transient transfection methods include, for examplewithout limitation, microinjection, electroporation or particlebombardment. Polynucleotides may be included in vectors, such as forexample plasmid vectors or viral vectors.

Also provided herein are isolated cells and cell lines obtained by theabove-described methods of engineering cells provided herein. In someembodiments, an isolated cell comprises at least one CAR as describedabove. In some embodiments, an isolated cell comprises a population ofCARs, each CAR comprising different extracellular ligand-bindingdomains.

Also provided herein are isolated immune cells obtained according to anyone of the methods described above. Any immune cell capable ofexpressing heterologous DNAs can be used for the purpose of expressingthe CAR of interest. In some embodiments, the immune cell is a T cell.In some embodiments, an immune cell can be derived from, for examplewithout limitation, a stem cell. The stem cells can be adult stem cells,non-human embryonic stem cells, more particularly non-human stem cells,cord blood stem cells, progenitor cells, bone marrow stem cells, inducedpluripotent stem cells, totipotent stem cells or hematopoietic stemcells. Representative human cells are CD34+ cells. The isolated cell canalso be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell,a B-cell or a T cell selected from the group consisting of inflammatoryT-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes, memoryT-lymphocytes, or helper T-lymphocytes. In some embodiments, the cellcan be derived from the group consisting of CD4+ T-lymphocytes and CD8+T-lymphocytes.

Prior to expansion and genetic modification, a source of cells can beobtained from a subject through a variety of non-limiting methods. Cellscan be obtained from a number of non-limiting sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue, cordblood, thymus tissue, tissue from a site of infection, ascites, pleuraleffusion, spleen tissue, and tumors. In some embodiments, any number ofT cell lines available and known to those skilled in the art, may beused. In some embodiments, cells can be derived from a healthy donor,from a patient diagnosed with cancer or from a patient diagnosed with aninfection. In some embodiments, cells can be part of a mixed populationof cells which present different phenotypic characteristics.

Also provided herein are cell lines obtained from a transfected T cellaccording to any of the above-described methods. Also provided hereinare modified cells resistant to an immunosuppressive treatment. In someembodiments, an isolated cell according to the invention comprises apolynucleotide encoding a CAR.

The immune cells of the invention can be activated and expanded, eitherprior to or after genetic modification of the T cells, using methods asgenerally described, for example without limitation, in U.S. Pat. Nos.6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466;6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843;5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent ApplicationPublication No. 20060121005. T cells can be expanded in vitro or invivo. Generally, the T cells of the invention can be expanded, forexample, by contact with an agent that stimulates a CD3 TCR complex anda co-stimulatory molecule on the surface of the T cells to create anactivation signal for the T cell. For example, chemicals such as calciumionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogeniclectins like phytohemagglutinin (PHA) can be used to create anactivation signal for the T cell.

In some embodiments, T cell populations may be stimulated in vitro bycontact with, for example, an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, a population of T cells can becontacted with an anti-CD3 antibody and an anti-CD28 antibody, underconditions appropriate for stimulating proliferation of the T cells.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-Vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2,IL-15, TGFp, and TNF, or any other additives for the growth of cellsknown to the skilled artisan. Other additives for the growth of cellsinclude, but are not limited to, surfactant, plasmanate, and reducingagents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media caninclude RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 10, and X-Vivo20, Optimizer, with added amino acids, sodium pyruvate, and vitamins,either serum-free or supplemented with an appropriate amount of serum(or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂). T cells that havebeen exposed to varied stimulation times may exhibit differentcharacteristics

In some embodiments, the cells of the invention can be expanded byco-culturing with tissue or cells. The cells can also be expanded invivo, for example in the subject's blood after administering the cellinto the subject.

In some embodiments, an isolated cell according to the present inventioncomprises one inactivated gene selected from the group consisting ofCD52, dCK, GR, PD-1, CTLA-4, LAG3, Tim3, BTLA, BY55, TIGIT, B7H5, LAIR1,SIGLEC10, 2B4, HLA, TCRα and TCRβ and/or expresses a CAR, a multi-chainCAR and/or a pTα transgene. In some embodiments, an isolated cellcomprises polynucleotides encoding polypeptides comprising a multi-chainCAR. In some embodiments, the isolated cell according to the presentinvention comprises two inactivated genes selected from the groupconsisting of: CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, GR and TCRα,GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 andTCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ,TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 andTCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 andTCRα, 2B4 and TCRβ and/or expresses a CAR, a multi-chain CAR and a pTαtransgene.

In some embodiments, TCR is rendered not functional in the cellsaccording to the invention by inactivating TCRα gene and/or TCRβgene(s). In some embodiments, a method to obtain modified cells derivedfrom an individual is provided, wherein the cells can proliferateindependently of the major histocompatibility complex (MHC) signalingpathway. Modified cells, which can proliferate independently of the MHCsignaling pathway, and which may be obtained by this method areencompassed in the scope of the present invention. Modified cellsdisclosed herein can be used for treating patients in need thereofagainst Host versus Graft (HvG) rejection and Graft versus Host Disease(GvHD); therefore in the scope of the present invention is a method oftreating patients in need thereof against Host versus Graft (HvG)rejection and Graft versus Host Disease (GvHD) comprising treating saidpatient by administering to said patient an effective amount of modifiedcells comprising inactivated TCRα and/or TCRβ genes.

In some embodiments, the immune cells are engineered to be resistant toone or more chemotherapy drugs. The chemotherapy drug can be, forexample, a purine nucleotide analogue (PNA), thus making the immune cellsuitable for cancer treatment combining adoptive immunotherapy andchemotherapy. Exemplary PNAs include, for example, clofarabine,fludarabine, and cytarabine, alone or in combination. PNAs aremetabolized by deoxycytidine kinase (dCK) into mono-, di-, andtri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNAsynthesis, act as pro-apoptotic agents, and are potent inhibitors ofribonucleotide reductase (RNR), which is involved in trinucleotideproduction. Provided herein are EGFRvIII specific CAR-T cells comprisingan inactivated dCK gene. In some embodiments, the dCK knockout cells aremade by transfection of T cells using polynucleotides encoding specificTAL-nuclease directed against dCK genes by, for example, electroporationof mRNA. The dCK knockout EGFRvIII specific CAR-T cells are resistant toPNAs, including for example clorofarabine and/or fludarabine, andmaintain T cell cytotoxic activity toward EGFRvIII-expressing cells. Inanother example, the chemotherapy drug can be, for example, aCD52-targeting molecule, such as a monoclonal anti-CD52 antibody (e.g.alemtuzumab). CD52 is a protein present on the surface of lymphocytes,and anti-CD52 antibodies can induce apoptosis and lysis of immune cellsthrough antibody- and complement-dependent cytotoxicity, leading tolymphodepletion. Provided herein are EGFRvIII specific CAR-T cellscomprising an inactivated CD52 gene. In some embodiments, CD52 knockoutcells are made by transfection of T cells using polynucleotides encodinga specific TAL-nuclease directed against the CD52 gene by, for example,electroporation of mRNA. The CD52 knockout EGFRvIII specific CAR-T cellsare resistant to anti-CD52 molecules, including for example alemtuzumab,and maintain T cell cytotoxic activity toward EGFRvIII-expressing cellsin the presence of anti-CD52 molecules.

In some embodiments, isolated cells or cell lines of the invention cancomprise a pTα or a functional variant thereof. In some embodiments, anisolated cell or cell line can be further genetically modified byinactivating the TCRα gene.

In some embodiments, the CAR-T cell comprises a polynucleotide encodinga suicide polypeptide, such as for example RQR8. See, e.g.,WO2013153391A, which is hereby incorporated by reference in itsentirety. In CAR-T cells comprising the polynucleotide encoding asuicide polypeptide, the suicide polypeptide is expressed at the surfaceof a CAR-T cell. In some embodiments, the suicide polypeptide comprisesthe amino acid sequence shown in SEQ ID NO: 226.CPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCSGGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPRPW (SEQ ID NO. 226).

In some embodiments, the suicide polypeptide may also comprise a signalpeptide at the amino terminus. When the suicide polypeptide is expressedat the surface of a CAR-T cell, binding of rituximab to the R epitopes[i.e. the epitope recognized by rituximab-CPYSNPSLC (SEQ ID NO: 256)])of the polypeptide causes lysis of the cell. More than one molecule ofrituximab may bind per polypeptide expressed at the cell surface. Each Repitope of the polypeptide may bind a separate molecule of rituximab.Deletion of EGFRvIII specific CAR-T cells may occur in vivo, for exampleby administering rituximab to a patient. The decision to delete thetransferred cells may arise from undesirable effects being detected inthe patient which are attributable to the transferred cells, such as forexample, when unacceptable levels of toxicity are detected.

In some embodiments, a suicide polypeptide may contain one, two, threeor more epitopes recognized by an antibody (e.g. rituximab).

In some embodiments, a suicide polypeptide may be provided in anEGFRvIII specific CAR T cell in a polypeptide that is separate from theCAR-containing polypeptide. In some embodiments, a suicide polypeptidemay be provided in an EGFRvIII specific CAR T cell in the samepolypeptide chain as the CAR polypeptide. In CARs containing a suicidepolypeptide, typically the suicide polypeptide is provided in theextracellular portion of the CAR.

In a CAR containing a suicide polypeptide, the suicide polypeptide maycontain, for example, one or more copies of the amino acid sequence ofthe epitope recognized by rituximab [CPYSNPSLC (SEQ ID NO: 256)]. Thesuicide polypeptide may be located at different positions in the CAR.For example, the suicide polypeptide may be N-terminal to the scFv or itmay be C-terminal to the scFv in the CAR. In some embodiments, thesuicide peptide may be between the scFv and the hinge region of the CAR.In some embodiments, a CAR may contain more than one suicidepolypeptide. For example, a CAR may contain a suicide polypeptideN-terminal to the scFv and a suicide polypeptide C-terminal to the scFv.Each of these polypeptides may contain one or more copies of the epitoperecognized by rituximab. For example, a CAR may contain a first suicidepolypeptide at a position N-terminal to the scFv, wherein the firstsuicide polypeptide contains one copy of the epitope recognized byrituximab, and a second suicide polypeptide at a position C-terminal tothe scFv, wherein the second suicide polypeptide contains two copies ofthe epitope recognized by rituximab.

Also provided herein are nucleic acids encoding EGFRvIII-specific CARsthat contain a suicide polypeptide sequence in the CAR.

In an example, a suicide polypeptide in the same polypeptide chain as anEGFRvIII specific CAR may have the sequence provided described herein asthe “R2 suicide sequence”. The R2 suicide sequence contains two copiesof the epitope recognized by rituximab [CPYSNPSLC (SEQ ID NO: 256)].Such EGFRvIII-specific CARs may be referred to herein as “EGFRvIII-R2CARs”. Table 50 provides amino acid sequences of exemplary EGFRvIII-R2CARs of the present invention. In Table 5D, the signal/leader peptidesequence is in bold, the GS linker [(GGGGS)₄ (SEQ ID NO: 202)] isunderlined, and the R2 suicide sequence is in bold and underlined. Table5E provides exemplary nucleic acid sequences encoding exemplaryEGFRvIII-R2 CARs of the present invention.

TABLE 5D  Amino acid sequences of exemplary EGFRvIII specific CARs with R2 suicide sequence Components (in order, N-terminus CARCAR Amino Add Sequence to C-terminus) 14C11-MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETL CD8α signal peptide; R2TLTCTVSGFSLNNARMGVSWIRQPPGKALEWFAHIFSTD 14C11 VH (Table 1, SEQEKSFRTSLRSRLTLSKDTSKSQVVLTMTNMDPVDTATYY ID NO: 15);CARDSSNYEGYFDYWGQGILVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEIVMTQSPATLSVSPGERATLSCRASQSVSNNL 14C11 VL (Table 1, SEQAWYQQKPGQAPRLLIYGASTRATGVPARFSGSDSGTEFS ID NO: 16);LTISSLQSEDFAVYFCQQYKDWPFTFGPGTKVEIK

R2 suicide sequence;

TTTPAP CD8α hinge; RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8α TM;IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF 4-1BB signalingMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA domain;YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR CD3zeta signalingKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY domain QGLSTATKDTYDALHMQALPPR(SEQ ID NO: 248) 32A10- MALPVTALLLPLALLLHAARPQVTLKESGPVLVKPTETLCD8α signal peptide; R2 TLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSTD32A10 VH (Table 1 SEQ EKSIRRSLRSRLTLSKDTSKSQVVLTMTNMDPVDTATYFID NO: 11); CARDSSNYEGYFDYWGQGTLVTVSSGGGGSGGGGSGGGG GS linker;SGGGGSEVVMTQSPATLSVSPGERVTLSCRASQSVSSNF 32A10 VL (Table 1 SEQAWYQQRPGQAPRLLLYGATTRATGLPGRFSGSGSGTENI ID NO: 12);LTISSLQSEDFAIYFCQQYKDWPFTFGPGSKVDIK

R2 suicide sequence;

TTTPAP CD8α hinge; RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8α TM;IYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF 4-1BB signalingMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA domain;YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR CD3zeta signalingKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY domainQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 249) 26B9-R2MALPVTALLLPLALLLHAARPEVQLVESWGVLVKPGGSL CD8α signal peptide;RLSCAASGFIFNNAWMSWVRQAPGKGLEWIGRIKSKSDG 26B9 VH (Table 1, SEQGTTDYAAPVKDRFTISRDDSKDTLYLQMNGLKTEDTAVY ID NO: 41);FCTTAPGGPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSG GS linker;GGGSDIVLTQSPLSLPVTPGEPASISCRSSQSLLHRDGF 26B9 VL (Table 1, SEQNYLDWFLQKPGQSPQLLIYLASSRASGVPDRFSGSDSGT ID NO: 42);DFTLKISRVEAEDVGVYYCMQALQTPITFGQGTRLEIK R2 suicide sequence;

TT CD8α hinge; TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD CD8α TM;FACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF 4-1BB signalingKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSA domain;DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG CD3zeta signalingKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH domainDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 250) 30D8-R2MALPVTALLLPLALLLHAARPEVQTLVESGGGLVKPGGSL CD8α signal peptide;RLSCEASGFTFSDAWMSWVRQAPGKGLEWVGRIKSKTDG 30D8 VH (Table 1, SEQGTTDYVVPLNGRFIISRDDSRNTLYLQLNNLKTEDTAVY ID NO: 37);YCTTVPGSYGYWGQGTLVTVSSGGGGSGGGGSGGGGSGG GS linker;GGSDIVMTQSPLSLPVTPGEPASISCRSSQSLLHNKRNN 30D8 VL (Table 1, SEQYLDWFLQKPGQSPQLLIYLASNRASGVPDRFSGGGSGTD ID NO: 38);FTLKISRVEAEDVGVYYCMQAQQTPITFGQGTRLEIK

R2 suicide sequence;

TTTP CD8α hinge; APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CD8α TM;CDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQ 4-1BB signalingPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADA domain;PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP CD3zeta signalingRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG domainLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 251)

TABLE 5E  Nucleic acid sequences of exemplary EGFR VIII specific CARs with R2 suicide sequence CAR CAR Nucleic Add Sequence Components 14C11-ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTG CD8α signal peptide; R2CTGCTGCACGCAGCCAGACCCCAGGTGACACTGAAGGAGAG 14C11 VH (Table 1, SEQCGGCCCCGTGCTGGTGAAGCCTACAGAGACACTGACCCTGA ID NO: 15);CCTGCACAGTGAGCGGCTTCTCCCTGAACAATGCAAGGATG GS linker;GGCGTGTCCTGGATCAGGCAGCCACCTGGCAAGGCCCTGGA 14C11 VL (Table 1, SEQGTGGTTCGCCCACATCTTTAGCACCGACGAGAAGTCCTTTCG ID NO: 16);CACATCTCTGAGAAGCAGGCTGACCCTGAGCAAGGATACAA R2 suicide sequence;GCAAGTCCCAGGTGGTGCTGACCATGACAAACATGGACCCT CD8α hinge;GTGGATACCGCCACATACTATTGTGCCCGGGACAGCTCCAAT CD8α TM;TACGAGGGCTATTTCGATTACTGGGGCCAGGGCATCCTGGT 4-1BB signalingGACCGTGTCTAGCGGCGGCGGCGGCTCTGGAGGAGGAGGA domain;AGCGGAGGAGGAGGATCCGGCGGCGGCGGCTCTGAGATCG CD3zeta signalingTGATGACCCAGTCCCCAGCCACACTGTCTGTGAGCCCAGGA domainGAGAGAGCCACCCTGTCTTGCAGGGCCTCCCAGTCTGTGAGCAACAATCTGGCCTGGTATCAGCAGAAGCCTGGCCAGGCCCCAAGGCTGCTGATCTACGGAGCAAGCACCAGAGCAACAGGAGTGCCTGCAAGGTTCTCCGGATCTGACAGCGGCACCGAGTTTTCTCTGACAATCTCCTCTCTGCAGAGCGAGGACTTCGCCGTGTATTTTTGTCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCACAAAGGTGGAGATCAAGGGCTCCGGAGGAGGAGGATCCTGCCCCTATTCCAACCCTTCTCTGTGCAGCGGAGGAGGAGGAAGCTGTCCATACTCCAATCCCTCCCTGTGCTCCGGCGGCGGAGGATCCACCACAACCCCAGCACCTAGACCACCAACCCCAGCACCAACAATCGCATCCCAGCCTCTGTCTCTGCGGCCCGAGGCATGCAGGCCAGCAGCAGGCGGCGCCGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTATTGCAAGCGCGGCCGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTCGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGCGCGTGAAGTTTTCCCGGTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGAAGAGATCCCGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGTCCACCGCCACAAAGGACACCTACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA (SEQ ID NO: 252) 32A10-ATGGCACTGCCAGTGACCGCCCTGCTGCTGCCTCTGGCCCTG CD8α signal peptide; R2CTGCTGCACGCAGCCAGACCCCAGGTGACACTGAAGGAGTC 32A10 VH (Table 1 SEQCGGCCCCGTGCTGGTGAAGCCTACAGAGACACTGACCCTGA ID NO: 11);CCTGCACAGTGAGCGGCTTCTCTCTGAGCAACGCAAGGATG GS linker;GGCGTGTCCTGGATCAGGCAGCCACCTGGCAAGGCCCTGGA 32A10 VL (Table 1 SEQGTGGCTGGCCCACATCTTTTCCACCGACGAGAAGTCTATCCG ID NO: 12);GAGAAGCCTGCGCTCCCGGCTGACCCTGAGCAAGGATACAT R2 suicide sequence;CCAAGTCTCAGGTGGTGCTGACCATGACAAACATGGACCCT CD8α hinge;GTGGATACCGCCACATACTTCTGTGCCCGGGACAGCTCCAAT CD8α TM;TACGAGGGCTATTTTGATTACTGGGGCCAGGGCACCCTGGT 4-1BB signalingGACAGTGTCTAGCGGAGGAGGAGGAAGCGGAGGAGGAGG domain;ATCAGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGAGGTG CD3zeta signalingGTCATGACCCAGTCTCCAGCCACACTGAGCGTGTCCCCAGGA domainGAGCGCGTGACCCTGAGCTGCCGGGCCTCTCAGAGCGTGTCCTCTAACTTCGCCTGGTATCAGCAGCGGCCCGGACAGGCACCAAGGCTGCTGCTGTACGGAGCAACCACAAGAGCAACAGGCCTGCCTGGCAGGTTTTCCGGCTCTGGCAGCGGCACCGAGAATATCCTGACAATCAGCTCCCTGCAGAGCGAGGACTTCGCCATCTATTTTTGTCAGCAGTACAAGGATTGGCCATTCACCTTTGGCCCCGGCTCCAAGGTGGACATCAAGGGATCCGGAGGAGGAGGATCTTGCCCCTATTCTAACCCTAGCCTGTGCTCCGGAGGAGGAGGATCCTGTCCATACTCTAATCCATCCCTGTGCAGCGGAGGAGGAGGATCTACCACAACCCCAGCACCTAGACCACCAACCCCAGCACCCACAATCGCCTCTCAGCCTCTGAGCCTGCGCCCAGAGGCATGCAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTCGCCTGCGATATCTATATCTGGGCACCACTGGCAGGAACCTGTGGCGTGCTGCTGCTGTCCCTGGTCATCACCCTGTATTGCAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGGCGGCTGTGAGCTGAGAGTGAAGTTTTCCAGGTCTGCCGATGCCCCAGCCTATCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGAGGAGAGGACGCGATCCCGAGATGGGAGGCAAGCCTAGGCGCAAGAACCCACAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACCGCCACAAAGGACACCTACGATGCCCTGCACATGCAGGCCCTGCCTCCAAGGTGA (SEQ ID NO: 253) 26B9-R2ATGGCCCTGCCAGTGACCGCCCTGCTGCTGCCACTGGCCCTG CD8α signal peptide;CTGCTGCACGCCGCCAGACCTGAGGTGCAGCTGGTGGAGAG 26B9 VH (Table 1, SEQCTGGGGCGTGCTGGTGAAGCCAGGAGGCTCTCTGAGGCTG ID NO: 41);AGCTGCGCAGCATCCGGCTTCATCTTTAACAATGCCTGGATG GS linker;TCCTGGGTGAGACAGGCACCAGGCAAGGGCCTGGAGTGGA 26B9 VL (Table 1, SEQTCGGCAGGATCAAGAGCAAGTCCGACGGAGGAACCACAGA ID NO: 42);TTACGCAGCACCCGTGAAGGACCGCTTCACAATCTCTCGGGA R2 suicide sequence;CGATAGCAAGGATACCCTGTATCTGCAGATGAACGGCCTGA CD8α hinge;AGACAGAGGACACCGCCGTGTACTTCTGCACCACAGCCCCA CD8α TM;GGCGGCCCCTTTGATTATTGGGGCCAGGGCACACTGGTGAC 4-1BB signalingCGTGAGCTCCGGAGGAGGAGGAAGCGGCGGAGGAGGCAG domain;CGGCGGCGGCGGCTCTGGCGGCGGCGGCAGCGACATCGTG CD3zeta signalingCTGACACAGAGCCCACTGTCCCTGCCTGTGACCCCAGGAGA domainGCCCGCCTCTATCAGCTGTCGCTCTAGCCAGAGCCTGCTGCACCGGGACGGCTTCAATTACCTGGATTGGTTTCTGCAGAAGCCTGGCCAGAGCCCACAGCTGCTGATCTATCTGGCCTCCTCTAGAGCATCCGGAGTGCCTGACAGGTTCTCCGGATCTGACAGCGGCACAGACTTCACCCTGAAGATCTCCCGCGTGGAGGCAGAGGATGTGGGCGTGTACTATTGCATGCAGGCCCTGCAGACACCAATCACCTTCGGCCAGGGCACACGGCTGGAGATCAAGGGATCCGGAGGAGGAGGATCTTGCCCCTACTCTAACCCTAGCCTGTGCAGCGGCGGAGGAGGATCTTGTCCATATTCTAATCCAAGCCTGTGCAGCGGGGGAGGAGGAAGCACCACAACCCCTGCACCAAGACCCCCTACACCAGCACCTACCATCGCATCCCAGCCACTGTCTCTGCGGCCCGAGGCATGTAGGCCAGCAGCAGGAGGAGCAGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTCAAGCAGCCTTTTATGCGCCCAGTGCAGACAACCCAGGAGGAGGACGGCTGCTCCTGTAGGTTCCCAGAAGAGGAGGAGGGAGGATGTGAGCTGCGCGTGAAGTTTTCCCGGTCTGCCGATGCACCTGCATACCAGCAGGGACAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGACGCGATCCTGAGATGGGAGGCAAGCCCCGGAGAAAGAACCCTCAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATTCCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCTACAGCCACCAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCACCAA GGTGA (SEQ ID NO: 254) 30D8-R2ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCTCTGGCCCTG CD8α signal peptide;CTGCTGCACGCAGCCAGACCAGAGGTGCAGCTGGTGGAGTC 30D8 VH (Table 1, SEQCGGAGGAGGCCTGGTGAAGCCAGGAGGCTCCCTGAGGCTG ID NO: 37);TCTTGCGAGGCCAGCGGCTTCACCTTTAGCGACGCCTGGAT GS linker;GTCCTGGGTGAGACAGGCACCAGGCAAGGGCCTGGAGTGG 30D8 VL (Table 1, SEQGTGGGCAGGATCAAGAGCAAGACAGACGGCGGCACCACAG ID NO: 38);ATTACGTGGTGCCTCTGAACGGCCGGTTCATCATCTCCCGCG R2 suicide sequence;ACGATTCTCGGAATACCCTGTATCTGCAGCTGAACAATCTGA CD8α hinge;AGACAGAGGATACCGCCGTGTACTATTGCACCACAGTGCCT CD8α TM;GGCTCCTACGGCTATTGGGGCCAGGGCACACTGGTGACCGT 4-1BB signalingGAGCTCCGGCGGCGGCGGCTCTGGAGGAGGAGGAAGCGG domain;AGGAGGAGGAAGCGGGGGCGGCGGCTCTGACATCGTGATG CD3zeta signalingACACAGTCTCCACTGAGCCTGCCAGTGACCCCAGGAGAGCC domainTGCCTCCATCTCTTGTCGCTCTAGCCAGTCCCTGCTGCACAACAAGCGGAACAATTACCTGGATTGGTTCCTGCAGAAGCCAGGCCAGTCTCCCCAGCTGCTGATCTATCTGGCCAGCAATAGAGCCTCCGGAGTGCCAGACAGGTTCTCTGGAGGAGGAAGCGGAACAGACTTCACCCTGAAGATCAGCCGCGTGGAGGCAGAGGACGTGGGCGTGTACTATTGCATGCAGGCCCAGCAGACACCCATCACCTTTGGCCAGGGAACCCGGCTGGAGATCAAGGGCTCCGGAGGAGGAGGATCCTGCCCTTACTCCAACCCATCTCTGTGCAGCGGAGGAGGAGGATCTTGTCCATATTCCAATCCTTCCCTGTGCTCCGGAGGAGGAGGAAGCACCACAACCCCTGCACCAAGACCCCCTACACCAGCACCTACCATCGCATCCCAGCCTCTGTCTCTGCGGCCCGAGGCATGTAGGCCAGCAGCAGGCGGCGCCGTGCACACCAGGGGCCTGGACTTTGCCTGCGATATCTACATCTGGGCACCACTGGCAGGAACATGTGGCGTGCTGCTGCTGTCTCTGGTCATCACCCTGTACTGCAAGAGAGGCAGGAAGAAGCTGCTGTATATCTTCAAGCAGCCCTTCATGCGGCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCTGAAGAGGAGGAGGGAGGATGTGAGCTGCGCGTGAAGTTTAGCCGGTCCGCCGATGCACCAGCATACCAGCAGGGCCAGAACCAGCTGTATAACGAGCTGAATCTGGGCCGGAGAGAGGAGTACGACGTGCTGGATAAGAGGAGGGGACGCGATCCTGAGATGGGAGGCAAGCCTCGGAGAAAGAACCCACAGGAGGGCCTGTACAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTATAGCGAGATCGGCATGAAGGGAGAGAGGCGCCGGGGCAAGGGACACGATGGCCTGTACCAGGGCCTGTCCACAGCCACCAAGGACACCTATGATGCCCTGCACATGCAGGCCCTGCCACCAAGGTGA (SEQ ID NO: 255)

Therapeutic Applications

Isolated cells obtained by the methods described above, or cell linesderived from such isolated cells, can be used as a medicament. In someembodiments, such a medicament can be used for treating cancer,including solid tumors and liquid tumors. In some embodiments, thecancer is EGFRvIII related cancer (e.g., any EGFRvIII expressing cancer)including, but not limited to, glioblastoma (e.g., glioblastomamultiform), anaplastic astrocytoma, giant cell glioblastoma,gliosarcoma, anaplastic oligodendroglioma, anaplastic ependymoma,anaplastic oligoastrocytoma, choroid plexus carcinoma, anaplasticganglioglioma, pineoblastoma, pineocytoma, meningioma,medulloepithelioma, ependymoblastoma, medulloblastoma, supraentorialprimitive neuroectodermal tumor, atypical teratoid/rhabdoid tumor, mixedglioma, head and neck cancer, non-small cell lung cancer, breast cancer,ovarian cancer, prostate cancer, medullobastoma, colorectal cancer, analcancer, cervical cancer, renal cancer, skin cancer, pancreatic cancer,liver cancer, bladder cancer, gastric cancer, thyroid cancer,mesothelioma, uterine cancer, lymphoma, or leukemia.

In some embodiments, the medicament as described herein can be usedfor 1) inhibiting tumor growth or progression in a subject who hasmalignant cells expressing EGFRvIII; 2) inhibiting metastasis ofmalignant cells expressing EGFRvIII in a subject; and 3) inducing tumorregression in a subject who has malignant cells expressing EGFRvIII.

In some embodiments, an isolated cell according to the invention, orcell line derived from the isolated cells, can be used in themanufacture of a medicament for treatment of a cancer in a patient inneed thereof.

Also provided herein are methods for treating patients. In someembodiments the method comprises providing an immune cell of theinvention to a patient in need thereof. In some embodiments, the methodcomprises a step of administering transformed immune cells of theinvention to a patient in need thereof.

In some embodiments, T cells of the invention can undergo robust in vivoT cell expansion and can persist for an extended amount of time.

Methods of treatment of the invention can be ameliorating, curative orprophylactic. The method of the invention may be either part of anautologous immunotherapy or part of an allogenic immunotherapytreatment. The invention is particularly suitable for allogeneicimmunotherapy. T cells from donors can be transformed intonon-alloreactive cells using standard protocols and reproduced asneeded, thereby producing CAR-T cells which may be administered to oneor several patients. Such CAR-T cell therapy can be made available as an“off the shelf” therapeutic product.

Cells that can be used with the disclosed methods are described, forexample, in the previous section. Treatment can be used to treatpatients diagnosed with, for example, cancer. Cancers that may betreated include, for example, cancers that are related to EGFRvIII,including any of the above-listed cancers. Types of cancers to betreated with the CARs and CAR-T cells of the invention include, but arenot limited to certain liquid and solid tumors, such as glioblastomamultiforme, head and neck cancer, non-small cell lung cancer, breastcancer, ovarian cancer and prostate cancer. Adult tumors/cancers andpediatric tumors/cancers are also included.

In some embodiments, the treatment can be in combination with one ormore therapies against cancer selected from the group of antibodiestherapy, antibody-drug conjugate therapy, chemotherapy, targetedtherapy, cytokines therapy, vaccine therapy, oncolytic virus therapy,dendritic cell therapy, gene therapy, nanoparticle therapy, hormonetherapy, surgical resection, laser light therapy, tumor treating fields,and radiation therapy. For example, CARs and CAR-T cells of theinvention can be administered to a patient in conjunction with (e.g.,before, simultaneously, or following) 1) standard of care, includingradiation, surgical resection, chemotherapy (e.g., temozolomide,procarbazine, carmustine, lomustine, vincristine etc.), antibody therapysuch as bevacizumab, anti-angiogenic therapy, and/or tumor treatingfields; 2) vaccine, including EGFRvIII vaccine; 3) antibody-drugconjugate therapy, including but not limited to drug conjugates thattarget the HER family of receptors such as EGFR, HER2, HER3, and HER4;4) targeted therapy, such as kinase inhibitors (e.g., everolimus); and5) immunotherapies, including but not limited to anti-PD-1, anti-PD-L1,anti-PD-L2, anti-41BB, anti-TIM3, anti-LAG3, anti-TIGIT, anti-OX40,anti-HVEM, anti-BTLA, anti-CD40, anti-CD47, anti-CSF1R, anti-CSF1,anti-MARCO, anti-IL8, anti-CXCR4, and anti-CTLA4 antibodies.

In some embodiments, treatment can be administered to patientsundergoing an immunosuppressive treatment. Indeed, the inventionpreferably relies on cells or population of cells, which have been maderesistant to at least one immunosuppressive agent (e.g., cyclosporin,azathioprine, methotrexate, mycophenolate, and FK506) due to theinactivation of a gene encoding a receptor for such immunosuppressiveagent. In this aspect, the immunosuppressive treatment should help theselection and expansion of the T cells according to the invention withinthe patient. The administration of the cells or population of cellsaccording to the invention may be carried out in any convenient manner,including by aerosol inhalation, injection, ingestion, transfusion,implantation or transplantation. The compositions described herein maybe administered to a patient subcutaneously, intradermally,intratumorally, intracranially, intranodally, intramedullary,intramuscularly, by intravenous or intralymphatic injection, orintraperitoneally. In one embodiment, the cell compositions of theinvention are preferably administered by intravenous injection.

In some embodiments, treatment can be administered to patientsundergoing a lymphodepletion regimen. Depletion of the immune regulatoryelements with cytotoxic agents or whole body irradiation can enhance theanti-tumor activity of the CARs and CAR-T cells of the presentinvention. For example, the cytotoxic agent in a lymphodepletion regimenincludes, but is not limited to, fludarabine, cyclophosphamide, and/oralemtuzumab.

In some embodiments the administration of the cells or population ofcells can comprise the administration of, for example, about 10⁴ toabout 10⁹ cells per kg body weight including all integer values of cellnumbers within those ranges. In some embodiments the administration ofthe cells or population of cells can comprise the administration ofabout 10⁵ to 10⁸ cells per kg body weight including all integer valuesof cell numbers within those ranges. The cells or population of cellscan be administered in one or more doses. In some embodiments, saideffective amount of cells can be administered as a single dose. In someembodiments, said effective amount of cells can be administered as morethan one dose over a period time. Timing of administration is within thejudgment of a managing physician and depends on the clinical conditionof the patient. The cells or population of cells may be obtained fromany source, such as a blood bank or a donor. While individual needsvary, determination of optimal ranges of effective amounts of a givencell type for a particular disease or conditions within the skill of theart. An effective amount means an amount which provides a therapeutic orprophylactic benefit. The dosage administered will be dependent upon theage, health and weight of the recipient, kind of concurrent treatment,if any, frequency of treatment and the nature of the effect desired. Insome embodiments, an effective amount of cells or composition comprisingthose cells are administered parenterally. In some embodiments,administration can be an intravenous administration. In someembodiments, administration can be directly done by injection within atumor.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include one or more containers comprising a polynucleotideencoding an EGFRvIII specific CAR, or an engineered immune cellcomprising a polynucleotide encoding EGFRvIII specific CAR as describedherein, and instructions for use in accordance with any of the methodsof the invention described herein. Generally, these instructionscomprise a description of administration of the engineered immune cellfor the above described therapeutic treatments.

The instructions relating to the use of the engineered immune cells asdescribed herein generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso acceptable.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an EGFRvIII antibody. The container may further comprisea second pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Incorporated by reference herein for all purposes is the content of U.S.Provisional Patent Application Nos. 62/281,533 (filed Jan. 21, 2016) and62/431,758 (Filed Dec. 8, 2016).

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the invention in any way. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and fall within the scope of the appendedclaims.

EXAMPLES Example 1: Affinity Determination for Recombinant Anti-EGFRvIIIMurine-Human Chimeric Antibody and Humanized Antibodies

This example determines the affinity of chimeric and humanizedanti-EGFRvIII antibodies at 25° C. and 37° C.

Anti-EGFRvIII mouse (m) antibody, m62G7, generated from hybridomas wassequenced and subcloned into suitable vectors for expression asmurine-human chimeric antibodies. The CDRs of mouse antibody m62G7 weregrafted onto human framework and expressed as human IgG1 recombinantantibody, h62G7. Affinity variants of h62G7 were made by introducingmutations in the CDRs of the heavy and light chains. The affinities ofrecombinant anti-EGFRvIII chimeric antibody m62G7 and humanized h62G7antibodies were measured on a surface plasmon resonance Biacore™ T200biosensor equipped with a research-grade anti-human Fc coupled CM4sensor chip (GE Healthcare Inc., Piscataway, N.J.). Anti-EGFRvIIIantibodies were then captured by anti-human Fc. Monomeric 8-histidinetagged human EGFRvIII extracellular domain was then injected as theanalyte at 10-fold dilution series with top concentration at 1000 nM.Affinity of anti-EGFRvIII antibodies towards human EGFRvIII was measuredat both 25° C. and 37° C. (Table 6). None of these antibodies showeddetectable binding to 1000 nM 8-histidine tagged recombinant wild-typeprotein EGFRwt under the same assay condition.

In Table 6, variants of h62G7 are described with reference to the heavychain variation then the light chain variation. For example, antibodyclone “h62G7-EQ/L6” refers to the h62G7 clone containing the “EQ”variation in the heavy chain (also referred to herein as “h62G7-EQ”) andthe “L6” variation in the light chain (also referred to herein as“h62G7-L6”). These heavy chain and light chain amino acid sequences areprovided in Table 2. Also, in the present application, a h62G7 variantmay be referred to with either the heavy chain or the light chainvariant written first—so, for example, “h62G7-EQ/L6” and “h62G7-L6/EQ”both refer to an antibody which contains a h62G7-EQ heavy chain and ah62G7-L6 light chain.

TABLE 6 25° C. 37° C. Antibody k_(a)(1/Ms) k_(d)(1/s) K_(D)(nM)k_(a)(1/Ms) k_(d)(1/s) K_(D)(nM) m62G7 7.30E+05 6.40E−02 88.7 8.00E+051.70E−01 207.0 h62G7-EQ/L6 2.40E+05 1.00E−02 43.8 6.60E+05 7.40E−02112.8 h62G7-EQ/L1-DV 2.00E+05 1.20E−05 59.9 3.70E+05 6.90E−02 185.8h62G7-H14/L1-DV 1.80E+04 2.00E−02 1087.9 6.60E+04 1.00E−01 1539.6h62G7-H14/L6 1.30E+04 1.30E−02 992.2 4.30E+04 6.80E−02 1583.3

Example 2: Affinity Determination for Human Anti-EGFRvIII Antibodies

This example determines the affinity of various human anti-EGFRvIIIantibodies at 37° C.

To generate human antibodies against EGFRvIII, transgenic AlivaMab mice(Ablexis LLC, San Francisco, Calif.) were immunized with alternatingschedule of paraformaldehyde-fixed rat glioblastoma cell line expressingEGFRvIII, F98-npEGFRvIII (American Type Culture Collection, Manassas,Va.) and peptides (SEQ ID NO: 227: CGSGSGLEEKKGNYWTDH) directed to thejunction region in EGFRvIII. Hybridomas were generated using standardtechniques. To determine the binding affinity and specificity of thesehybridomas to EGFRvIII, antibodies in culture supernatants were capturedby anti-mouse Fc using Biacore™ T200 biosensor equipped with anti-mouseFc coupled CM4 sensor chips (Biacore™ AB, Uppsala, Sweden—now GEHealthcare). Monomeric 8-histidine tagged human EGFRvIII extracellulardomain was then injected as the analyte at 10-fold dilution seriesstarting with top concentration 1000 nM. Affinity of anti-EGFRvIIIantibodies towards human EGFRvIII was measured at 37° C. (Table 7). Noneof these hybridoma antibodies showed detectable binding to 1000 nM8-histidine tagged recombinant wild-type protein EGFRwt under the sameassay condition.

TABLE 7 EGFRvIII binding at 37° C. Antibody k_(a) (1/Ms) k_(d) (1/s)K_(D) (nM) 42G9 6.88E+04 5.63E−04 8.2 32A10 6.54E+04 6.26E−04 9.6 21E116.66E+04 6.32E−04 9.5 49B11 7.64E+04 6.95E−04 9.1 46E10 5.97E+047.16E−04 12.0  12H6 5.93E+04 7.33E−04 12.4  19A9 5.58E+04 1.04E−03 18.6 11B11 5.21E+04 1.13E−03 21.7  21E7 6.52E+04 1.30E−03 19.9  20B9 4.67E+041.50E−03 32.1  12B2 7.38E+04 1.79E−03 24.3  11F10 6.63E+04 2.81E−0342.4  17G11 5.61E+04 3.00E−03 53.5  29D5 1.02E+05 4.24E−03 41.6  14C117.55E+04 5.93E−03 78.5  20E12 3.99E+04 1.41E−02 353.4  20G5 1.25E+052.89E−02 231.2  26B9 1.31E+05 3.20E−02 244.3  30D8 1.61E+05 2.77E−02172.0  32G8 6.82E+03 1.22E−02 1788.9   34E7 3.77E+04 1.28E−02 339.5 

Example 3: Binding Specificity of Anti-EGFRvIII Antibodies to EGFRvIIIExpressing Cell Lines by Flow Cytometry

This example demonstrates the cell binding specificity of anti-EGFRvIIIantibodies to EGFRvIII expressing cells.

To assess the cell binding specificity of anti-EGFRvIII antibodiesgenerated from the AlivaMab mice, three isogenic rat glioblastoma celllines and a human cancer cell line were used: F98 (does not express anyform of human EGFR), F98-EGFRwt (expresses wild-type EGFR),F98-npEGFRvIII (expresses EGFRvIII) and A431 (an epidermoid carcinomacell line with wild-type EGFR over-expression), all obtained fromAmerican Type Culture Collection (Manassas, Va.). For cell staining,500,000 cells were incubated with 50 μl hybridoma supernatants for 45min at 4° C., washed with binding buffer (PBS (Phosphate BufferedSaline)+0.5% BSA (Bovine Serum Albumin)), followed by incubation withFITC-conjugated goat anti-mouse Fc specific secondary antibody fromJackson ImmunoResearch Laboratories (West Grove, Pa.). Tables 8A and 8Bshow mean fluorescent intensities (MFI) of EGFRvIII antibodies (exceptclone 20G5) on EGFRvIII expressing cell line were at least 10-foldhigher than on non-expressing cell lines. FIG. 1A, FIG. 1B, and FIG. 1Cshow examples of the FACS binding histograms of three EGFRvIII specificclones which had been cloned and expressed as recombinant human IgG1antibodies, 42G9 (FIG. 1A), 32A10 (FIG. 1B) and 32G8 (FIG. 1C), to thethree F98 cell lines.

TABLE 8A F98 F98-EGFRwt F98-EGFRvIII A431 % % % % Antibody MFI positiveMFI positive MFI positive MFI positive 2nd Ab only 170 0.6 202 1.7 2582.3 592 0.4 anti-EGFR(wt 163 0.5 9608 98.3 5329 99.4 55240 100.0 andvIII) 42G9 159 0.4 185 1.6 3247 98.5 538 0.3 32A10 159 0.5 185 1.4 334998.3 531 0.2 21E11 159 0.3 184 1.3 3105 98.5 555 0.5 49B11 156 0.6 1851.3 2980 98.5 599 0.8 46E10 158 0.4 187 1.6 2986 98.7 560 0.5 12H6 1570.5 188 1.9 3445 98.3 569 0.8 19A9 158 0.5 168 1.6 3100 98.1 578 1.011B11 161 0.6 187 1.7 3391 98.2 589 1.2 21E7 159 0.3 184 1.3 3105 98.5603 1.1 20B9 157 0.3 189 1.8 3418 98.3 558 0.7 12B2 156 0.4 185 1.5 274997.9 571 0.8 11F10 155 0.5 187 1.6 3283 98.0 582 1.1 17G11 157 0.6 1841.5 3357 98.1 556 0.7 29D5 155 0.3 185 1.3 2829 97.9 531 0.4 14C11 1570.4 185 1.3 3213 98.2 580 0.8

TABLE 8B F98 F98-EGFRwt F98-EGFRvIII A431 % % % % Antibody MFI positiveMFI positive MFI positive MFI positive 2nd Ab only 235 0.2 252 0.2 3221.3 185 0.7 anti-EGFR(wt 245 0.3 6857 97.2 5827 99.4 44493 100.0 andvIII) 20E12 381 6.0 348 3.4 3976 97.9 302 2.6 20G5 1248 16.8 1070 12.64639 98.5 391 2.0 26B9 310 4.1 298 2.3 5405 98.6 276 1.7 30D8 296 4.0280 1.7 5165 98.6 269 1.3 32G8 329 4.9 301 1.6 3734 98.6 271 1.2 34E7485 6.9 371 4.0 4128 98.5 294 1.1

These results demonstrate the binding specificity of the anti-EGFRvIIIantibodies to cells that express EGFRvIII.

Example 4: Affinity Determination for Fully Human Anti-EGFRvIIIAntibodies from Phage Library

This example determines the affinity of various human anti-EGFRvIIIantibodies at 25° C.

Human anti-EGFRvIII antibodies were sequenced and subcloned intosuitable vectors for expression as recombinant human IgG1 antibodies.The affinities of antibodies were measured at 25° C. (Table 9) on asurface plasmon resonance Biacore™ T200 biosensor equipped with ananti-human Fc coupled CM4 sensor chip (GE Healthcare Inc., Piscataway,N.J.). Anti-EGFRvIII antibodies were captured by anti-human Fc.Monomeric 8-histidine tagged human EGFRvIII extracellular domain wasthen injected as the analyte at 10-fold dilution series starting at 1000nM. Among the two antibodies, only C6 showed very weak but detectablebinding to 1000 nM 8-histidine tagged recombinant wild-type proteinEGFRwt at 25° C.

TABLE 9 EGFRvIII binding at 25° C. Antibody k_(a) (1/Ms) k_(d) (1/s)K_(D) (nM) B5 2.08E+04 1.41E−02 677.9 C6 1.68E+04 8.94E−03 532.1

Example 5: Affinity Determination for Recombinant Single Chain FvFormatted Anti-EGFRvIII Antibodies

This example determines the affinity of various recombinant single chainFv formatted anti-EGFRvIII antibodies at 37° C.

To convert conventional antibody into single chain Fv (scFv) fragment,the heavy chain variable domain and the light chain variable domain werejoined together via a (GGGGS)₄ (SEQ ID NO: 202) linker. The scFvfragment was then fused to human IgG1 Fc moiety to facilitaterecombinant expression and purification. Affinities of the scFv-Fcfusion proteins were measured at 37° C. on a surface plasmon resonanceBiacore™ T200 biosensor equipped with an anti-human Fc coupled CM4sensor chip (GE Healthcare Inc., Piscataway, N.J.) as described above.scFv-Fc proteins were captured by anti-human Fc. Monomeric 8-histidinetagged human EGFRvIII extracellular domain was then injected as theanalyte at 10-fold dilution series starting at 1000 nM. Table 10 showsthat scFv reformatted fusion proteins retain binding to EGFRvIII andthat the affinities of the scFv-Fc proteins in both HL (with theheavy-chain variable domain at the N-terminus) and LH (with thelight-chain variable domain at the N-terminus) orientations arecomparable to their conventional antibody counterparts listed in Tables6, 7 and 9. These scFv-Fc proteins were also tested for binding to 1000nM 8-histidine tagged recombinant wild-type protein EGFRwt at 25° C.,but none of them showed significant binding.

TABLE 10 EGFRvIII binding at 37° C. Antibody ka (1/Ms) kd (1/s) KD (nM)h62G7-L6/EQ 7.5E+05 7.6E−02 100.9  C6.HL 1.9E+04 2.0E−02 1063.8  14C11.HL 4.2E+04 4.3E−03 104.3  14C11.LH 4.8E+04 6.5E−03 136.0  20B9.HL2.4E+04 1.8E−03 73.8  20B9.LH 3.0E+04 2.5E−03 82.7  32A10.HL 4.7E+041.2E−03 24.5  32A10.LH 4.1E+04 1.2E−03 29.8  42G9.HL 5.6E+04 1.1E−0318.8  42G9.LH 4.0E+04 8.8E−04 22.1  26B9.HL 5.7E+04 4.0E−02 704.9 26B9.LH 6.9E+04 3.4E−02 494.2  30D8.HL 2.3E+05 5.3E−02 224.6  30D8.LH1.1E+05 3.8E−02 348.6  20E12.HL 2.1E+04 1.9E−02 893.7  20E12.LH 1.6E+041.5E−02 932.5  32G8.HL 8.7E+03 1.3E−02 1490.0  

Example 6: Production and Detection of EGFRvIII Specific ChimericAntigen Receptor (CAR) T Cells

This example determines the expression and antigen binding specificityof EGFRvIII specific CAR T cells.

PBMC from healthy donors provided by AllCells (Alameda, Calif.) werethawed according to the provider's specification and cultured overnightin X-Vivo™-15 medium (Lonza, Walkersville, Md.) supplemented with 5%Human serum. T cells were activated for 3 days in X-Vivo™-15 medium(Lonza) supplemented with 20 ng/mL Human IL-2, 5% Human serum, andDynabeads Human T activator CD3/CD28 at a bead:cell ratio 1:1 (LifeTechnologies, Carlsbad, Calif.). T cells were then transduced with abicistronic lentiviral vector harboring a BFP-T2A-EGFRvIII specific CARexpression cassette under the control of the EF1a promoter at amultiplicity of infection (MOI) of 5. In this construct, the EGFRvIIIspecific CAR is co-expressed with BFP (blue fluorescent protein), tofacilitate detection of the EGFRvIII specific CAR. The EGFRvIII specificCARs contained VH and VL sequences of different anti-EGRRvIII clones(h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6, 20E12, 2689, 3008, and 32G8;described elsewhere herein). CAR T cells were maintained in culture forup to 14 days post-transduction. Percentage of cells expressing theEGFRvIII specific CAR was monitored over time (on Day 4, 9, and 14post-lentivirus transduction of primary T cells) by flow cytometry usingBFP (blue fluorescent protein) for detection (FIG. 2A) (determined bypercentage of BFP positive viable cells).

To determine target binding specificity of the CARs, recombinantproteins EGFRvIII-mFc and EGFR-mFc, which comprise of the extracellulardomain of either human EGFRvIII or human wild-type EGFR, respectively,fused with mouse IgG1-Fc domain, were produced in HEK293 cells. Thetarget binding specificity was determined by incubating the differentEGFRvIII specific CAR T cells with either EGFRvIII-mFc or EGFR-mFcprotein followed by a PE-labeled goat anti-mouse Fc secondary antibody(Jackson ImmunoResearch) and analysed by flow cytometry on Day 4 posttransduction of the T cells with vectors encoding CARs containingdifferent EGFRvIII specific clones (h62G7-L6/EQ, 14C11, 2089, 32A10,42G9, C6, 20E12, 26B9, 30D8, and 32G8). As shown in FIG. 28, CAR T cellscomprising the VH and VL sequences of clones h62G7-L6/EQ, 14C11, 20B9,32A10, 42G9, 20E12, 2689, and 30D8 bind to EGFRvIII-mFc but do notsignificantly bind to EGFR-mFc. On Day 14 post-transduction, thecorrelation of CAR expression as detected by BFP on the cells andrecombinant EGFRvIII-mFc binding by the cells was determined. Arepresentative set of data is shown in FIG. 2C, which shows theexpression of CARs containing ten different EGFRvIII specific clones(h62G7-L6/EQ, 14C11, 2089, 32A10, 42G9, C6, 20E12, 26B9, 30D8, and 32G8)in CAR T cells on Day 14 post-transduction, as determined by BFPdetection on the cells and recombinant EGFRvIII-mFc binding by thecells.

These results demonstrate that CAR T cells containing CARs comprisingthe VH and VL sequences of clones h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9,20E12, 26B9, and 30D8 bind to EGFRvIII-mFc but do not significantly bindto EGFR-mFc.

Example 7: Target Dependent and Independent Degranulation of EGFRvIIISpecific CAR T Cells

This example determines the degranulation activity of EGFRvIII specificCAR T cells in the presence and absence of target-expression cancer celllines.

Five cell lines were used to evaluate the degranulation activity of CART cells. Human lung cancer cell line NCI-H522 and glioblastoma cell lineU87MG were obtained from American Type Culture Collection (Manassas,Va.) and cultured in RPMI 1640 medium supplemented with 10% heatinactivated fetal bovine serum (Mediatech Inc., Manassas, Va.). Sincemost cancer cell lines do not express EGFRvIII, NCI-H522 (which does notexpress detectable level of wild-type EGFR or EGFRvIII) was transducedwith a lentivirus vector encoding the full length EGFRvIII gene (SEQ IDNO: 201) to generate “NCI-H522-EGFRvIII”, which expresses low level ofEGFRvIII. To generate isogenic human glioblastoma cell lines thatexpress various EGFR proteins, the endogenous wild-type EGFR gene ofU87MG was first knocked out to generate an EGFR-null cell line “U87-KO”.U87-KO cell was then transduced with a lentivirus vector encoding thefull length wild-type EGFR gene (accession number NP_005219) to obtainEGFR overexpressing cell line, “U87-KO-EGFRwt”. Similarly,“U87-KO-EGFRvIII” (which over-expresses EGFRvIII), was generated fromU87-KO by lentivirus transduction with a vector encoding the full lengthEGFRvIII gene (SEQ ID NO: 201).

For the degranulation assay, 100,000 T-cells expressing various EGFRvIIIspecific CARs (containing clones h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9,C6, 20E12, 26B9, 30D8, and 32G8) were incubated in 96-well plates withan equal number of cancer cells expressing various levels of theEGFRvIII protein or EGFRwt protein. Co-cultures were maintained in afinal volume of 100 μl of X-Vivo™-15 medium (Lonza, Walkersville, Md.)for 6 hours at 37° C. with 5% CO₂. CD107a staining was done during cellstimulation, by the addition of a fluorescent anti-CD107a antibody(Miltenyi Biotec, San Diego, Calif.) at the beginning of the co-culture,together with a final concentration of 1 μg/ml of anti-CD49d (BDPharmingen, San Diego, Calif.), 1 μg/ml of anti-CD28 (Miltenyi Biotec),and 1× Monensin (eBioscience, San Diego, Calif.) solution. After the 6 hincubation period, cells were stained with a fixable viability dye andfluorochrome-conjugated anti-CD8 antibody (Miltenyi Biotec) and analyzedby flow cytometry. The degranulation activity was determined as the % ofviable/CD8+/BFP+/CD107a+ cells, and by determining the mean fluorescenceintensity signal (MFI) for CD107a staining among CD8+/BFP+ cells.Degranulation assays were carried out on Day 14 after CAR transduction.FIG. 3 shows the results from two PBMC donors for each EGFRvIII specificCAR. Except for CAR T cells containing clones C6 and 32G8, an increasein degranulation activity (over CAR-T alone) was only observed whenEGFRvIII specific CAR T cells were cocultured with cell lines thatexpress EGFRvIII, such as NCI-H522-EGFRvIII and U87-KO-EGFRvIII.

These results demonstrate that EGFRvIII specific CAR T cells exhibitdegranulation activity in the presence of EGFRvIII-expressing tumorcells, but do not exhibit degranulation activity in the presence oftumor cells that do not express EGFRvIII.

Example 8: Interferon Gamma (IFNγ) Secretion of EGFRvIII Specific CAR TCells

This example shows the level of IFNγ secretion of EGFRvIII specific CART cells upon co-culture of EGFRvIII specific CAR T cells with targetprotein expressing cell lines.

EGFRvIII specific CAR T-cells expressing CARs containing differentEGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6,20E12, 26B9, 30D8, and 32G8) were incubated in 96-well plates (100,000cells/well), together with an equal number of cells expressing variouslevels of EGFRvIII or EGFRwt proteins (cells NCI-H522,NCI-H522-EGFRvIII, U87-KO, U87-KO-EGFRwt, and U87-KO-EGFRvIII; each aredescribed above in Example 7). Co-cultures were maintained in a finalvolume of 100 μl of X-Vivo™-15 medium (Lonza) for 18 hours at 37° C.with 5% CO₂. The supernatant was then collected and frozen. The IFNγ inthe supernatant was measured with the Human IFN-gamma Quantikine ELISAKit (R&D systems, Minneapolis, Minn.) according to the manufacturer'sspecifications. As shown in FIG. 4, for the majority of CAR T cells, thelevel of IFNγ secretion correlates with the amount of EGFRvIII expressedby the co-cultured cells: low expressor NCI-H522-EGFRvIII induced smallamount of IFNγ, high expressor U87-KO-EGFRvIII induced large amount ofIFNγ. T cells expressing EGFRvIII specific clone 32G8 CAR did notsecrete significant levels of IFNγ under all conditions tested. EGFRvIIIspecific clone C6 CAR T cells, on the other hand, secreted high levelsof IFNγ upon coculture with either U87-KO-EGFRwt or U87-KO-EGFRvIIIcells.

These results demonstrate that for the majority of EGFRvIII specific CART cells, the level of IFNγ secretion correlates with the amount ofEGFRvIII expressed by co-cultured cells

Example 9: Cytotoxicity of EGFRvIII Specific CAR T Cells

This example determines the cytotoxicity of EGFRvIII specific CAR Tcells upon co-culture of EGFRvIII specific CAR T cells with targetprotein expressing cell lines.

EGFRvIII specific CAR T-cells expressing CARs containing differentEGFRvIII specific clones (h62G7-L6/EQ, 14C11, 20B9, 32A10, 42G9, C6,20E12, 2669, 30D8, and 32G8) were seeded in 96-well plates (400,000cells/well), together with 20,000 target cells expressing various levelsof the EGFRvIII protein. The target cells were: U87-KO-EGFRwt,NCI-H522-EGFRvIII, and U87-KO-EGFRvIII, described above. Target(EGFRvIII positive: NCI-H522-EGFRvIII and U87-KO-EGFRvIII) and control(EGFRvIII negative: U87-KO-EGFRwt) cells were plated and labelled withthe fluorescent intracellular dye CFSE before co-culturing them with theEGFRvIII specific CAR T-cells. The co-cultures were incubated for 4hours at 37° C. with 5% CO₂. After this incubation period, cells werelabelled with a fixable viability dye and analyzed by flow cytometry.Viability of each cellular population (EGFRvIII positive target cells orEGFRvIII negative control cells) was determined and the % of specificcell lysis was calculated. Cytotoxicity assays were carried out 14 dayspost CAR transduction of the T cells. All EGFRvIII specific CAR T cells,except 32G8, were able to lyse both low-level target expressing cells(NCI-H522-EGFRvIII) and high-level target expressing cells(U87-KO-EGFRvIII) to various degrees. In addition, EGFRvIII specificclone C6 CAR T cells lysed both wild-type EGFR and EGFRvIII expressingcells (FIG. 5).

These results demonstrate that EGFRvIII-specific CAR T cells effectivelykill cells that express EGFRvIII.

Example 10: In Vivo Study of EGFRvIII Specific CAR T Cells inU87-KO-EGFRvIII Model

This example shows the anti-tumor activity of EGFRvIII specific CAR Tcells in a subcutaneous U87-KO-EGFRvIII GBM xenograph model.

Three million U87-KO-EGFRvIII GBM tumor cells (described above) wereimplanted subcutaneously into 5-6 week old NSG mice (Jackson Laboratory,Sacramento, Calif.). Tumor volume was measured once a week by a caliperdevice and calculated with the following formula: Tumorvolume=(length×width²)/2. On day 8 post tumor implantation, animals wererandomized by tumor sizes into five animals per group and a single doseof 6 million CAR positive EGFRvIII specific CAR T cells (expressing theEGFRvIII specific clone 14C11, 32A10 or 26B9; the clones are describedelsewhere herein) or the equivalent total number of non-transduced Tcells were administered through bolus tail vein injection. FIG. 6 showsthat the 14C11, 32A10 and 26B9 EGFRvIII specific CAR T cells, but notthe non-transduced T cells, inhibited U87-KO-EGFRvIII xenograft tumorgrowth in vivo.

These results demonstrate that EGFRvIII-specific CAR T cells effectivelyinhibit tumor growth of EGFRvIII-expressing cells in vivo.

Example 11: Surface Expression of EGFRvIII Specific CARs Containing theR2 Suicide Sequence

This example shows the expression of EGFRvIII specific CARs containingthe R2 suicide sequence in T cells.

The R2 suicide sequence is a CD20 epitope based suicide sequence andcontains two tandem copies of the rituximab recognition epitope. The R2sequence was inserted between the scFv and the CD8a hinge sequences ofvarious EGFRvIII specific CARs described elsewhere herein (14C11, 32A10,30D8, and 26B9) to generate various EGFRvIII-R2 CARs (referred to hereinas “14C11-R2”, “32A10-R2”, “30D8-R2”, and “26B9-R2”, respectively). Tcells were transduced with a lentiviral vector harboring the variousEGFRvIII-R2 CAR expression cassettes under the control of the EF1apromoter at a multiplicity of infection (MOI) of 5 or 25 (for clones26B9-R2 and 30D8-R2). CAR T cells were maintained in culture for up to14 or 15 days post-transduction. CAR expression was monitored over time(on Day 4, Day 9/10, and Day 14/15 post-T cell transduction) by flowcytometry using biotinylated recombinant proteins: EGFRvIII-mFc orrituximab to the cells (conjugated with EZ-Link™ Sulfo-NHS-SS-Biotin;ThermoFisher, Waltham, Mass.), followed by PE conjugated streptavidin(BD Biosciences, San Diego, Calif.). The percentages of CAR positivecells as detected with biotinylated EGFRvIII-mFc and biotinylatedrituximab are shown in FIGS. 7 and 8, respectively. Binding tonon-transduced T cells (NTD) is provided as a control.

These results demonstrate the expression in T cells of EGFRvIII-specificCARs containing the R2 suicide sequence.

Example 12: Cytotoxicity of EGFRvIII-Specific R2 CAR T Cells

This example shows the cytotoxicity of EGFRvIII-specific R2 CAR T cellsupon co-culture with target expressing cell lines.

Cytotoxicity assays were carried out as described in Example 9, usingthe EGFRvIII-R2 CARs 14C11-R2, 32A10-R2, 30D8-R2, and 26B9-R2, which aredescribed in Example 11. All EGFRvIII specific R2 CAR T cells testedwere able to lyse both low-level target expressing cells(NCI-H522-EGFRvIII) and high-level target expressing cells(U87-KO-EGFRvIII) to various degrees (FIG. 9).

These results demonstrate that EGFRvIII-specific R2 CAR T cellseffectively kill cells that express EGFRvIII.

Although the disclosed teachings have been described with reference tovarious applications, methods, kits, and compositions, it will beappreciated that various changes and modifications can be made withoutdeparting from the teachings herein and the claimed invention below. Theforegoing examples are provided to better illustrate the disclosedteachings and are not intended to limit the scope of the teachingspresented herein. While the present teachings have been described interms of these exemplary embodiments, the skilled artisan will readilyunderstand that numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The foregoing description and Examples detail certain specificembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

1-32. (canceled)
 33. An isolated engineered immune cell comprising apolynucleotide encoding an Epidermal Growth Factor Receptor Variant III(EGFRvIII) specific chimeric antigen receptor (CAR), wherein thepolynucleotide is under the transcriptional control of a promoter,wherein the EGFR-specific CAR comprises an EGFRvIII-binding domain, atransmembrane domain, and an intracellular signaling domain, and whereinthe EGFRvIII-binding domain comprises: a) a heavy chain variable (VH)region comprising three complementarity determining regions of VHcomplementary determining region 1 (VH CDR1), VH complementarydetermining region 2 (VH CDR2), and VH complementary determining region3 (VH CDR3) of 42G9 arranged sequentially in the N terminus to Cterminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1comprises the amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2comprises the amino acid sequence of one of SEQ ID NOs: 77-78, and VHCDR3 comprises the amino acid sequence of SEQ ID NO: 79; and a lightchain variable (VL) region comprising three complementarity determiningregions of VL complementarity determining region 1 (VL CDR1), VLcomplementarity determining region 2 (VL CDR2), and VL complementaritydetermining region 3 (VL CDR3) of 42G9 arranged sequentially in the Nterminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3,wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 156, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 157, and VL CDR3comprises the amino acid sequence of SEQ ID NO: 158; or b) a VH regioncomprising three complementarity determining regions of VH CDR1, VHCDR2, and VH CDR3 of 32A10 arranged sequentially in the N terminus to Cterminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2comprises the amino acid sequence of one of SEQ ID NOs: 83-84, and VHCDR3 comprises the amino acid sequence of SEQ ID NO: 85; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 32A10 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 159, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 160, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 161; or c) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 20B9 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 86-87, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 79; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 20B9 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 162, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 164; or d) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 14C11 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 88-90, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 91-92, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 85; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 14C11 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 165, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 161; or e) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 30D8 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 109-111, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 112-113, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 114; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 30D8 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 182, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 183, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 184; or f) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 20E12 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 115-117, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 118-119, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 120; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 20E12 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 185, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 186, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 184; or g) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 26B9 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 121-123, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 124-125, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 126; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 26B9 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 187, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 188, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 189; or h) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of C6 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 137-139, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 140-141, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 142; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of C6 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 195, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 196, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 197; or i) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 12B2 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 102-103, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 104; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 12B2 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 176, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 172, and VL CDR3 comprises theamino acid sequence of SEQ ID NO:
 177. 34. The engineered immune cell ofclaim 33, wherein the EGFRvIII-binding domain is a single-chain variablefragment (scFv).
 35. The engineered immune cell of claim 34, wherein thescFv comprises a VH region and a VL region selected from the groupconsisting of: i) the VH region comprising the amino acid sequence ofSEQ ID NO: 9 and the VL region comprising the amino acid sequence of SEQID NO: 10 (42G9); ii) the VH region comprising the amino acid sequenceof SEQ ID NO: 11 and the VL region comprising the amino acid sequence ofSEQ ID NO: 12 (32A10); iii) the VH region comprising the amino acidsequence of SEQ ID NO: 13 and the VL region comprising the amino acidsequence of SEQ ID NO: 14 (20B9); iv) the VH region comprising the aminoacid sequence of SEQ ID NO: 15 and the VL region comprising the aminoacid sequence of SEQ ID NO: 16 (14C11); v) the VH region comprising theamino acid sequence of SEQ ID NO: 37 and the VL region comprising theamino acid sequence of SEQ ID NO: 38 (30D8) vi) the VH region comprisingthe amino acid sequence of SEQ ID NO: 39 and the VL region comprisingthe amino acid sequence of SEQ ID NO: 40 (20E12); vii) the VH regioncomprising the amino acid sequence of SEQ ID NO: 41 and the VL regioncomprising the amino acid sequence of SEQ ID NO: 42 (26B9); viii) the VHregion comprising the amino acid sequence of SEQ ID NO: 48 and the VLregion comprising the amino acid sequence of SEQ ID NO: 49 (C6); and ix)the VH region comprising the amino acid sequence of SEQ ID NO: 30 andthe VL region comprising the amino acid sequence of SEQ ID NO: 31(12B2).
 36. The engineered immune cell of claim 33, wherein the CARcomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 53-57 and 59-61.
 37. The engineered immune cell of claim 33,wherein the engineered immune cell expresses at its cell surfacemembrane the EGFRvIII-specific CAR.
 38. The engineered immune cell ofclaim 33, wherein the intracellular signaling domain comprises a CD3zetasignaling domain.
 39. The engineered immune cell of claim 33, whereinthe intracellular signaling domain is a first intracellular signalingdomain and the CAR comprises a second intracellular signaling domain.40. The engineered immune cell of claim 39, wherein the firstintracellular signaling domain comprises a CD3zeta signaling domain andthe second intracellular signaling domain comprises a 4-1BB signalingdomain.
 41. The engineered immune cell of claim 33, wherein theEGFRvIII-specific CAR comprises a second extracellular ligand-bindingdomain, wherein the second extracellular ligand-binding domain is notspecific for EGFRvIII.
 42. The engineered immune cell of claim 33,wherein the transmembrane domain of the EGFRvIII-specific CAR comprisesa transmembrane domain from the α chain of the high-affinity IgEreceptor (FcεRI), and wherein the engineered immune cell comprises asecond polynucleotide encoding a polypeptide comprising a transmembranedomain from the γ or β chain of FcεRI.
 43. The engineered immune cell ofclaim 33, wherein the engineered immune cell further comprises apolynucleotide encoding a CAR which is not specific for EGFRvIII. 44.The engineered immune cell of claim 33, wherein the engineered immunecell comprises a polynucleotide encoding a suicide polypeptide.
 45. Theengineered immune cell of claim 33, wherein the engineered immune cellcomprises a disruption of one or more endogenous genes selected from thegroup consisting of genes encoding TCRα, TCRβ, CD52, glucocorticoidreceptor (GR), deoxycytidine kinase (dCK), and programmed death-1(PD-1).
 46. The engineered immune cell of claim 33, wherein theengineered immune cell is obtained from a healthy donor.
 47. Theengineered immune cell of claim 33, wherein the engineered immune cellis obtained from a patient.
 48. The engineered immune cell of claim 33,wherein the engineered immune cell is derived from an inflammatoryT-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, amemory T-lymphocyte, a helper T-lymphocyte, a natural killerT-lymphocyte, or a natural killer cell.
 49. A method of engineering animmune cell comprising: introducing into an immune cell a polynucleotideencoding an EGFRvIII specific CAR, wherein the polynucleotide is underthe transcriptional control of a promoter, wherein the EGFR-specific CARcomprises an EGFRvIII-binding domain, a transmembrane domain, and anintracellular signaling domain, and wherein the EGFRvIII-binding domaincomprises: a) a heavy chain variable (VH) region comprising threecomplementarity determining regions of VH complementary determiningregion 1 (VH CDR1), VH complementary determining region 2 (VH CDR2), andVH complementary determining region 3 (VH CDR3) of 42G9 arrangedsequentially in the N terminus to C terminus direction of VH CDR1, VHCDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence ofone of SEQ ID NOs: 74-76, VH CDR2 comprises the amino acid sequence ofone of SEQ ID NOs: 77-78, and VH CDR3 comprises the amino acid sequenceof SEQ ID NO: 79; and a light chain variable (VL) region comprisingthree complementarity determining regions of VL complementaritydetermining region 1 (VL CDR1), VL complementarity determining region 2(VL CDR2), and VL complementarity determining region 3 (VL CDR3) of 42G9arranged sequentially in the N terminus to C terminus direction of VLCDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acidsequence of SEQ ID NO: 156, VL CDR2 comprises the amino acid sequence ofSEQ ID NO: 157, and VL CDR3 comprises the amino acid sequence of SEQ IDNO: 158; or b) a VH region comprising three complementarity determiningregions of VH CDR1, VH CDR2, and VH CDR3 of 32A10 arranged sequentiallyin the N terminus to C terminus direction of VH CDR1, VH CDR2, and VHCDR3, wherein VH CDR1 comprises the amino acid sequence of one of SEQ IDNOs: 80-82, VH CDR2 comprises the amino acid sequence of one of SEQ IDNOs: 83-84, and VH CDR3 comprises the amino acid sequence of SEQ ID NO:85; and a VL region comprising three complementarity determining regionsof VL CDR1, VL CDR2, and VL CDR3 of 32A10 arranged sequentially in the Nterminus to C terminus direction of VL CDR1, VL CDR2, and VL CDR3,wherein VL CDR1 comprises the amino acid sequence of SEQ ID NO: 159, VLCDR2 comprises the amino acid sequence of SEQ ID NO: 160, and VL CDR3comprises the amino acid sequence of SEQ ID NO: 161; or c) a VH regioncomprising three complementarity determining regions of VH CDR1, VHCDR2, and VH CDR3 of 20B9 arranged sequentially in the N terminus to Cterminus direction of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1comprises the amino acid sequence of one of SEQ ID NOs: 80-82, VH CDR2comprises the amino acid sequence of one of SEQ ID NOs: 86-87, and VHCDR3 comprises the amino acid sequence of SEQ ID NO: 79; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 20B9 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 162, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 164; or d) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 14C11 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 88-90, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 91-92, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 85; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 14C11 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 165, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 163, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 161; or e) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 30D8 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 109-111, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 112-113, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 114; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 30D8 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 182, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 183, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 184; or f) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 20E12 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 115-117, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 118-119, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 120; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 20E12 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 185, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 186, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 184; or g) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of 26B9 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 121-123, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 124-125, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 126; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of 26B9 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 187, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 188, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 189; or h) a VH region comprisingthree complementarity determining regions of VH CDR1, VH CDR2, and VHCDR3 of C6 arranged sequentially in the N terminus to C terminusdirection of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprisesthe amino acid sequence of one of SEQ ID NOs: 137-139, VH CDR2 comprisesthe amino acid sequence of one of SEQ ID NOs: 140-141, and VH CDR3comprises the amino acid sequence of SEQ ID NO: 142; and a VL regioncomprising three complementarity determining regions of VL CDR1, VLCDR2, and VL CDR3 of C6 arranged sequentially in the N terminus to Cterminus direction of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1comprises the amino acid sequence of SEQ ID NO: 195, VL CDR2 comprisesthe amino acid sequence of SEQ ID NO: 196, and VL CDR3 comprises theamino acid sequence of SEQ ID NO: 197; or i) a VH region comprisingthree complementarity determining regions of VH CDR1 VH CDR2, and VHCDR3 of 12B2 arranged sequentially in the N terminus to C terminusdirection of VH CDR1 VH CDR2, and VH CDR3, wherein VH CDR1 comprises theamino acid sequence of one of SEQ ID NOs: 74-76, VH CDR2 comprises theamino acid sequence of one of SEQ ID NOs: 102-103, and VH CDR3 comprisesthe amino acid sequence of SEQ ID NO: 104; and a VL region comprisingthree complementarity determining regions of VL CDR1, VL CDR2, and VLCDR3 of 12B2 arranged sequentially in the N terminus to C terminusdirection of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprisesthe amino acid sequence of SEQ ID NO: 176, VL CDR2 comprises the aminoacid sequence of SEQ ID NO: 172, and VL CDR3 comprises the amino acidsequence of SEQ ID NO: 177, thereby generating the engineered immunecell.
 50. A pharmaceutical composition comprising the engineered immunecell of claim
 33. 51. A method of treating a condition associated withmalignant cells expressing EGFRvIII in a subject comprisingadministering to a subject in need thereof an effective amount of thepharmaceutical composition of claim
 50. 52. The method of claim 51,wherein the condition is a cancer.